Antibodies to cd40 with enhanced agonist activity

ABSTRACT

Provided herein are agonistic antibodies, or antigen binding portions thereof, that bind to human CD40. Such antibodies optionally comprise Fc regions with enhanced specificity for FcγRIIb. The invention also provides methods of treatment of cancer or chronic infection by administering the antibodies of the invention to a subject in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.17/150,008, filed on Jan. 15, 2021, which is a Continuation of U.S.application Ser. No. 15/195,119, filed on Jun. 28, 2016, which claimspriority to U.S. Provisional Application No. 62/304,012, filed Mar. 4,2016. The contents of which are all incorporated herein by reference intheir entireties.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file SeqList_070413-20757.xml,created on Jul. 31, 2023 and containing 19,457 bytes.

BACKGROUND

Recent research has revealed that human cancers and chronic infectionsmay be treated with agents that modulate the patient's immune responseto malignant or infected cells. See, e.g., Reck & Paz-Ares (2015) Semin.Oncol. 42:402. Agonistic anti-CD40 antibodies, such as CP-870893 anddacetuzumab (SGN-40) have been tried for treating cancer based on thebelief that they may enhance such an immune response. See, e.g.,Kirkwood et al. (2012) CA Cancer J. Clin. 62:309; Vanderheide & Glennie(2013) Clin. Cancer Res. 19:1035. Recent experiments in mice haverevealed that anti-CD40 antibodies with enhanced specificity for theinhibitory Fc receptor FcγRIIb have increased anti-tumor efficacy. See,e.g., WO 2012/087928; Smith, et al. (2012) PNAS 109(16):6181-6; Li &Ravetch (2012) Proc. Nat'l Acad. Sci (USA) 109:10966; Wilson et al.(2011) Cancer Cell 19:101; White et al. (2011) J. Immunol. 187:1754.

The need exists for improved agonistic anti-human CD40 antibodies fortreatment of cancer and chronic infections in human subjects. Suchantibodies will preferably have enhanced specificity for the inhibitoryFc receptor FcγRIIb as compared to activating Fc receptors, and willexhibit enhanced anti-tumor and/or anti-infective activity.

SUMMARY OF THE INVENTION

Provided herein are isolated monoclonal antibodies (e.g., murinemonoclonal antibodies, humanized murine monoclonal antibodies, and humanmonoclonal antibodies) that specifically bind to human CD40 (the maturesequence of SEQ ID NO: 11), optionally having modified Fc regions thatenhance specificity for binding to FcγRIIb receptor. Also provided are(i) antibodies that compete with the antibodies disclosed herein forbinding to human CD40, and (ii) antibodies that bind to the sameepitopes as the antibodies disclosed herein, i.e. antibodies thatcompete for binding to human CD40 with antibodies comprising a mutant Fcregion having one or more mutations corresponding to one or moremutations in an IgG heavy chain selected from the group consisting ofN297A, S267E (“SE”), S267E/L328F (“SELF”), G237D/P238D/P271G/A330R(“V9”), or G237D/P238D/H268D/P271G/A330R (“V11”) (SEQ ID Nos: 3-7), suchas antibodies that compete for binding to human CD40 with mAb 2141 IgG1,also termed CP-870,893.

In some embodiments the antibody of the present invention comprises aheavy chain and a light chain, wherein the heavy chain comprises CDRH1,CDRH2 and CDRH3 sequences and the light chain comprises CDRL1, CDRL2 andCDRL3 and Fc variants N297A, S267E (“SE”), S267E/L328F (“SELF”),G237D/P238D/P271G/A330R (“V9”), G237D/P238D/H268D/P271G/A330R (“V11”),as disclosed at Table 2.

In some embodiments, FC variants include a N297A (SEQ ID Nos: 2,3), SE(SEQ ID Nos: 2,4), SELF (SEQ ID Nos: 2,5), V9 (SEQ ID Nos: 2,6), and orV11 (SEQ ID Nos: 2,7). In alternative embodiments, anti-human CD40antibodies of the present invention include antibodies comprising heavyand light chains consisting essentially of the sequences of these heavyand light chains, or comprise heavy and light chains sharing at least80%, 85%, 90% and 95% sequence identity with these sequences. In someembodiments, the anti-huCD40 antibodies of the present inventioncomprise modified Fc regions with greater specificity for binding toFcγRIIb as opposed to binding to activating receptors than antibodieswith naturally occurring Fc regions. In certain embodiments the A/Iratio for the anti-huCD40 antibody of the present invention is less than5, and in preferred embodiments, less than 1.

In certain embodiments, the invention relates to anti-huCD40 antibodiesor antigen binding fragments thereof that compete for binding with,cross-block, or bind to the same epitope as, one or more of antibodiescomprising Fc variants N297A (SEQ ID Nos: 2,3), SE (SEQ ID Nos: 2,4),SELF (SEQ ID Nos: 2,5), V9 (SEQ ID Nos: 2,6), and or V11 (SEQ ID Nos:2,7), including human or humanized antibodies.

In some embodiments the anti-huCD40 antibody of the present inventioncomprises one or more heavy chains and one or more light chains, such astwo heavy chains and two light chains.

In some embodiments the isolated antibody, or antigen binding portionthereof, that

-   -   (i) specifically binds to human CD40; and    -   (ii) comprises a mutant Fc region having one or more mutations        corresponding to one or more mutations in an IgG havey chain        selected from the group consisting of SEQ ID Nos: 3-7.

In some embodiments the isolated antibody or antigen binding portionthereof of competes for binding to human CD40 with CP-870,893 or ChiLob,2141.

In some embodiments the antibody or antigen binding portion thereof ofhas an enhanced specificity of binding to FcγRIIb.

The present invention further provides nucleic acids encoding the heavyand/or light chain variable regions, of the anti-CD40 antibodies of thepresent invention, or antigen binding fragments thereof, expressionvectors comprising the nucleic acid molecules, cells transformed withthe expression vectors, and methods of producing the antibodies byexpressing the antibodies from cells transformed with the expressionvectors and recovering the antibody.

The present invention also provides pharmaceutical compositionscomprising anti-huCD40 antibodies of the present invention, or antigenbinding fragments thereof, and a pharmaceutically acceptable carrier.

The present invention provides a method of enhancing an immune responsein a subject comprising administering an effective amount of ananti-huCD40 antibody of the present invention, or antigen bindingfragment thereof, to the subject such that an immune response in thesubject is enhanced. In certain embodiments, the subject has a tumor andan immune response against the tumor is enhanced. In another embodiment,the subject has a viral infection, e.g. a chronic viral infection, andan anti-viral immune response is enhanced.

The present invention also provides a method of inhibiting the growth oftumors in a subject comprising administering to the subject ananti-huCD40 antibody of the present invention, or antigen bindingfragment thereof, such that growth of the tumor is inhibited.

The present invention further provides a method of treating cancer,e.g., by immunotherapy, comprising administering to a subject in needthereof a therapeutically effective amount an anti-huCD40 antibody ofthe present invention, or antigen binding fragment thereof, e.g. as apharmaceutical composition, thereby treating the cancer. In certainembodiments, the cancer is bladder cancer, breast cancer,uterine/cervical cancer, ovarian cancer, prostate cancer, testicularcancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer,colorectal cancer, colon cancer, kidney cancer, head and neck cancer,lung cancer, stomach cancer, germ cell cancer, bone cancer, livercancer, thyroid cancer, skin cancer, neoplasm of the central nervoussystem, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancer.In certain embodiments, the cancer is a metastatic cancer, refractorycancer, or recurrent cancer.

In certain embodiments, the methods of modulating immune function andmethods of treatment described herein comprise administering ananti-huCD40 antibody of the present invention in combination with, or asa bispecific reagent with, one or more additional therapeutics, forexample, a second immunomodulatory antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C and 1D shows the characterization of CD40/FcγRhumanized mice and 2141 anti-CD40 Fc variants. FIG. 1A is arepresentative flow cytometry staining of mouse or human CD40 on theindicated splenic cell populations from CD40−/−huCD40+/+ and wild typemice. FIG. 1B is a representative flow cytometry staining of GC B cellsfrom mesenteric LN of the indicated mouse type. Live B220+ cells weregated. GC B cells indicated as CD38-Fashi. FIG. 1C illustrates an ELISAfor detection of serum levels of influenza H1N1-specific IgG from miceimmunized with recombinant influenza H1N1. Each dot representsindividual mouse. FIG. 1D illustrates the binding specificity of theindicated Fc variants of anti-CD40 Ab clone 2141 assessed by ELISA usingrecombinant hCD40. Data are represented as means. See also Table 1.

FIG. 2 shows the binding of hCD40 to mouse and human CD40L using SPRanalysis with immobilized human CD40 and soluble human/mouse CD40Ltitrated from 33 to 0.5 nM.

FIGS. 3A and 3B shows that human CD40 mAbs requires FcγR-engagement forin vivo activity. FIG. 3A shows FcγRs binding profile of 2141 anti-CD40Fc variants. See also Table 2.

FIG. 3B shows flow cytometry analysis for OVA-specific CD8+ T cells inthe blood of humanized CD40/FcγR mice immunized with DEC-OVA in thepresence or absence of the indicated CP-890,873 (left) or ChiLob 7/4(right) anti-CD40 Fc variants. Each dot represents an individual mouse.

FIGS. 4A, 4B, 4C and 4D shows increased activity of CP-870,893 byFc-engineering for FcγRIIB specific enhancement. FIG. 4A indicates thefold-change in hFcγRIIB and hFcγRIIB/hFcγRIIA^(R131) binding affinitiesof 2141 anti-CD40 Fc variants, based on SPR measurements. See also Table2. FIG. 4B shows flow cytometry analysis for OVA-specific CD8+ T cellsin the blood of huCD40/FcγR mice immunized with DEC-OVA in the presenceor absence of the indicated CP-870,893 anti-CD40 Fc variants. Each dotrepresents an individual mouse. FIG. 4C shows platelet counts in blood24 hours after administration of CP-870,893 anti-CD40 Fc variants intohumanized CD40/FcγR mice. Each dot represents individual mouse. FIG. 4Dshows hCD40⁺/hFcγRIIA⁺/hFcγRIIB⁺ or hCD40⁺/hFcγRIIA⁻/hFcγRIIB⁺ wereimmunized with DEC-OVA in the presence of CP-870,893-IgG2 and analyzedfor the percentages of OVA-specific CD8+ T cells in the blood at day 7.See also Table 2.

FIGS. 5A and 5B shows increased activity of CP-870,893 by Fc-engineeringfor FcγRIIB specific enhancement. FIG. 5A shows the change in total bodyweight over time after single injection of CP-870,893 Fc variant intohumanized FcγR/CD40 mice. Data is represented as mean+/−SEM. n=4. FIG.5B shows the platelet counts in humanized CD40/FcγR mice blood 7 daysafter administration of CP-870,893 anti-CD40 Fc variants. Each dotrepresents an individual mouse.

DETAILED DESCRIPTION

The present invention provides isolated antibodies, particularlymonoclonal antibodies, e.g., humanized or human monoclonal antibodies,that specifically bind to human CD40 (“huCD40”) and have agonistactivity. Sequences are provided for various humanized murineanti-huCD40 monoclonal antibodies. In certain embodiments, theantibodies described herein are derived from particular murine heavy andlight chain germline sequences and/or comprise particular structuralfeatures such as CDR regions comprising particular amino acid sequences.

Further provided herein are methods of making such antibodies,immunoconjugates and bispecific molecules comprising such antibodies orantigen-binding fragments thereof, and pharmaceutical compositionsformulated to contain the antibodies or fragments. Also provided hereinare methods of using the antibodies for immune response enhancement,alone or in combination with other immunostimulatory agents (e.g.,antibodies) and/or cancer or anti-infective therapies. Accordingly, theanti-huCD40 antibodies described herein may be used in a treatment in awide variety of therapeutic applications, including, for example,inhibiting tumor growth and treating chronic viral infections.

Definitions

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

CD40 refers to “TNF receptor superfamily member 5” (TNFRSF5). Unlessotherwise indicated, or clear from the context, references to CD40herein refer to human CD40 (“huCD40”), and anti-CD40 antibodies refer toanti-human CD40 antibodies. Human CD40 is further described at GENE IDNO: 958 and MIM (Mendelian Inheritance in Man): 109535. The sequence ofhuman CD40 (NP_001241.1), including 20 amino acid signal sequence, isprovided at SEQ ID NO: 11.

CD40 interacts with CD40 ligand (CD40L), which is also referred to asTNFSF5, gp39 and CD154. Unless otherwise indicated, or clear from thecontext, references to CD40L herein refer to human CD40L (“huCD40L”).Human CD40L is further described at GENE ID NO: 959 and MIM: 300386. Thesequence of human CD40L (NP_000065.1) is provided at SEQ ID NO: 12.

Unless otherwise indicated or clear from the context, the term“antibody” as used to herein may include whole antibodies and anyantigen-binding fragments (i.e., “antigen-binding portions”) or singlechains thereof. An “antibody” refers, in one embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen bindingfragment thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as V_(H)) and a heavy chain constantregion. In certain naturally occurring IgG, IgD and IgA antibodies, theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. In certain naturally occurring antibodies, each light chain iscomprised of a light chain variable region (abbreviated herein as V_(L))and a light chain constant region. The light chain constant region iscomprised of one domain, C_(L). The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four framework regions (FRs),arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁷ to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁶ M is generallyconsidered to indicate nonspecific binding. As used herein, an antibodythat “binds specifically” to an antigen refers to an antibody that bindsto the antigen and substantially identical antigens with high affinity,which means having a K_(D) of 10⁻⁷ M or less, preferably 10⁻¹⁰ M orless, even more preferably 5×10⁻⁹ M or less, and most preferably between10⁻⁸ M and 10⁻¹⁰ M or less, but does not bind with high affinity tounrelated antigens. An antigen is “substantially identical” to a givenantigen if it exhibits a high degree of sequence identity to the givenantigen, for example, if it exhibits at least 80%, at least 90%,preferably at least 95%, more preferably at least 97%, or even morepreferably at least 99% sequence identity to the sequence of the givenantigen. By way of example, an antibody that binds specifically to humanCD40 might also cross-react with CD40 from certain non-human primatespecies (e.g., cynomolgus monkey), but might not cross-react with CD40from other species, or with an antigen other than CD40.

Unless otherwise indicated, an immunoglobulin may be from any of thecommonly known isotypes, including but not limited to IgA, secretoryIgA, IgG and IgM. The IgG isotype is divided in subclasses in certainspecies: IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b andIgG3 in mice. Immunoglobulins, e.g., human IgG1, exist in severalallotypes, which differ from each other in at most a few amino acids.Unless otherwise indicated, “antibody” may include, by way of example,monoclonal and polyclonal antibodies; chimeric and humanized antibodies;human and non-human antibodies; wholly synthetic antibodies; and singlechain antibodies.

The term “antigen-binding portion” or “antigen binding fragment” of anantibody, as used herein, refers to one or more fragments of an antibodythat retain the ability to specifically bind to an antigen (e.g., humanCD40). Examples of binding fragments encompassed within the term“antigen-binding portion/fragment” of an antibody include (i) a Fabfragment—a monovalent fragment consisting of the V_(L), V_(H), C_(L) andCHI domains; (ii) a F(ab′)2 fragment—a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, and (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546)consisting of a V_(H) domain. An isolated complementarity determiningregion (CDR), or a combination of two or more isolated CDRs joined by asynthetic linker, may comprise and antigen binding domain of an antibodyif able to bind antigen.

Single chain antibody constructs are also included in the invention.Although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci.(USA) 85:5879-5883). Such single chain antibodies are also intended tobe encompassed within the term “antigen-binding portion/fragment” of anantibody. These and other potential constructs are described at Chan &Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. Antigen-binding portions/fragments can beproduced by recombinant DNA techniques, or by enzymatic or chemicalcleavage of intact immunoglobulins.

Unless otherwise indicated, the word “fragment” when used with referenceto an antibody, such as in a claim, refers to an antigen bindingfragment of the antibody, such that “antibody or fragment” has the samemeaning as “antibody or antigen binding fragment thereof.”

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs, giving rise totwo antigen binding sites with specificity for different antigens.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann (1990) Clin. Exp. Immunol. 79:315-321; Kostelnyet al. (1992)J. Immunol. 148, 1547-1553.

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope or a composition of antibodies in which all antibodies display asingle binding specificity and affinity for a particular epitope.Typically such monoclonal antibodies will be derived from a single cellor nucleic acid encoding the antibody, and will be propagated withoutintentionally introducing any sequence alterations. Accordingly, theterm “human monoclonal antibody” refers to a monoclonal antibody thathas variable and optional constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, human monoclonal antibodiesare produced by a hybridoma, for example, obtained by fusing a B cellobtained from a transgenic or transchromosomal non-human animal (e.g., atransgenic mouse having a genome comprising a human heavy chaintransgene and a light chain transgene), to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences are encoded by thegermline genes, but include subsequent rearrangements and mutations thatoccur, for example, during antibody maturation. As known in the art(see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid sequences that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not be identical to the original germline sequences,but instead will be substantially identical or similar (i.e., have atleast 80% identity).

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region also is derived from humangermline immunoglobulin sequences. Human antibodies of the presentinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody,” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. The terms “human” antibodies and “fully human”antibodies are used synonymously.

A “humanized” antibody refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody, e.g.a mouse antibody, are replaced with corresponding amino acids derivedfrom human immunoglobulins. In one embodiment of a humanized form of anantibody, some, most or all of the amino acids outside the CDR domainshave been replaced with amino acids from human immunoglobulins, whereassome, most or all amino acids within one or more CDR regions areunchanged. Small additions, deletions, insertions, substitutions ormodifications of amino acids are permissible as long as they do notabrogate the ability of the antibody to bind to a particular antigen. A“humanized” antibody retains an antigenic specificity similar to that ofthe original antibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody. A “hybrid” antibody refers to an antibodyhaving heavy and light chains of different types, such as a mouse(parental) heavy chain and a humanized light chain, or vice versa.

As used herein, “isotype” refers to the antibody class (e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that isencoded by the heavy chain constant region genes.

“Allotype” refers to naturally occurring variants within a specificisotype group, which variants differ in one or a few amino acids. See,e.g., Jefferis et al. (2009) mAbs 1:1.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody that binds specifically to an antigen.”

An “isolated antibody,” as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCD40 is substantially free of antibodies that specifically bind antigensother than CD40). An isolated antibody that specifically binds to anepitope of CD40 may, however, have cross-reactivity to other CD40proteins from different species.

“Effector functions,” deriving from the interaction of an antibody Fcregion with certain Fc receptors, include but are not necessarilylimited to C1q binding, complement dependent cytotoxicity (CDC), Fcreceptor binding, FcγR-mediated effector functions such as ADCC andantibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR).Such effector functions generally require the Fc region to be combinedwith an antigen binding domain (e.g., an antibody variable domain).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region ofan immunoglobulin. FcRs that bind to an IgG antibody comprise receptorsof the FcγR family, including allelic variants and alternatively splicedforms of these receptors. The FcγR family consists of three activating(FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA inhumans) and one inhibitory (FcγRIIb, or equivalently FcγRIIB) receptor.Various properties of human FcγRs are summarized in Table 1. Themajority of innate effector cell types co-express one or more activatingFcγR and the inhibitory FcγRIIb, whereas natural killer (NK) cellsselectively express one activating Fc receptor (FcγRIII in mice andFcγRIIIA in humans) but not the inhibitory FcγRIIb in mice and humans.Human IgG1 binds to most human Fc receptors and is considered equivalentto murine IgG2a with respect to the types of activating Fc receptorsthat it binds to.

TABLE 1 Properties of Human FcγRs Allelic Affinity for Fcγ variantshuman IgG Isotype preference Cellular distribution FcγRI None High IgG1= 3 > 4 >> 2 Monocytes, macrophages, activated described (K_(D)~10 nM)neutrophils, dendritic cells? FcγRIIA H131 Low to medium IgG1 > 3 > 2 >4 Neutrophils, monocytes, macrophages, R131 Low IgG1 > 3 > 4 > 2eosinophils, dendritic cells, platelets FcγRIIIA V158 Medium IgG1 = 3 >>4 > 2 NK cells, monocytes, macrophages, F158 Low IgG1 = 3 >> 4 > 2 mastcells, eosinophils, dendritic cells? FcγRIIb I232 Low IgG1 = 3 = 4 > 2 Bcells, monocytes, macrophages, T232 Low IgG1 = 3 = 4 > 2 dendriticcells, mast cells

An “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc”refers to the C-terminal region of the heavy chain of an antibody thatmediates the binding of the immunoglobulin to host tissues or factors,including binding to Fc receptors located on various cells of the immunesystem (e.g., effector cells) or to the first component (C1q) of theclassical complement system. Thus, an Fc region comprises the constantregion of an antibody excluding the first constant region immunoglobulindomain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fcregion comprises CH2 and CH3 constant domains in each of the antibody'stwo heavy chains; IgM and IgE Fc regions comprise three heavy chainconstant domains (CH domains 2-4) in each polypeptide chain. For IgG,the Fc region comprises immunoglobulin domains Cγ2 and Cγ3 and the hingebetween Cγ1 and Cγ2. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition C226 (e.g., SEQ ID NO: 14) or P230 (e.g., SEQ ID NO: 15) (or anamino acid between these two amino acids) to the carboxy-terminus of theheavy chain, wherein the numbering is according to the EU index as inKabat. Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, National Institutes of Health, Bethesda, MD; see also FIGS.3c-3f of U.S. Pat. App. Pub. No. 2008/0248028. The CH2 domain of a humanIgG Fc region extends from about amino acid 231 to about amino acid 340,whereas the CH3 domain is positioned on C-terminal side of a CH2 domainin an Fc region, i.e., it extends from about amino acid 341 to aboutamino acid 447 of an IgG (including a C-terminal lysine). As usedherein, the Fc region may be a native sequence Fc, including anyallotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc).Fc may also refer to this region in isolation or in the context of anFc-comprising protein polypeptide such as a “binding protein comprisingan Fc region,” also referred to as an “Fc fusion protein” (e.g., anantibody or immunoadhesin).

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally occurring variants thereof. Nativesequence Fc includes the various allotypes of Fcs. See, e.g., Jefferiset al. (2009) mAbs 1:1.

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., huCD40) to which an immunoglobulin or antibodyspecifically binds. Epitopes within protein antigens can be formed bothfrom contiguous amino acids (usually a linear epitope) or noncontiguousamino acids juxtaposed by tertiary folding of the protein (usually aconformational epitope). Epitopes formed from contiguous amino acids aretypically, but not always, retained on exposure to denaturing solvents,whereas epitopes formed by tertiary folding are typically lost ontreatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation.

The term “epitope mapping” refers to the process of identification ofthe molecular determinants on the antigen involved in antibody-antigenrecognition. Methods for determining what epitopes are bound by a givenantibody are well known in the art and include, for example,immunoblotting and immunoprecipitation assays, wherein overlapping orcontiguous peptides from (e.g., from CD40) are tested for reactivitywith a given antibody (e.g., anti-CD40 antibody); x-ray crystallography;2-dimensional nuclear magnetic resonance; yeast display; and HDX-MS(see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66, G. E. Morris, Ed. (1996)).

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same segment of aminoacid residues, as determined by a given method. Techniques fordetermining whether antibodies bind to the “same epitope on CD40” withthe antibodies described herein include, for example, epitope mappingmethods, such as, x-ray analyses of crystals of antigen:antibodycomplexes, which provide atomic resolution of the epitope, andhydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methodsmonitor the binding of the antibody to antigen fragments (e.g.proteolytic fragments) or to mutated variations of the antigen whereloss of binding due to a modification of an amino acid residue withinthe antigen sequence is often considered an indication of an epitopecomponent, such as alanine scanning mutagenesis (Cunningham & Wells(1985) Science 244:1081) or yeast display of mutant target sequencevariants. In addition, computational combinatorial methods for epitopemapping can also be used. These methods rely on the ability of theantibody of interest to affinity isolate specific short peptides fromcombinatorial phage display peptide libraries. Antibodies having thesame or closely related V_(L) and V_(H) or the same CDR sequences areexpected to bind to the same epitope.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that inhibit (partially or completely) the bindingof the other antibody to the target. Whether two antibodies compete witheach other for binding to a target, i.e., whether and to what extent oneantibody inhibits the binding of the other antibody to a target, may bedetermined using known competition experiments. In certain embodiments,an antibody competes with, and inhibits binding of another antibody to atarget by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.The level of inhibition or competition may be different depending onwhich antibody is the “blocking antibody” (i.e., the cold antibody thatis incubated first with the target). Competition assays can be conductedas described, for example, in Ed Harlow and David Lane, Cold SpringHarb. Protoc.; 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “UsingAntibodies” by Ed Harlow and David Lane, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind tothe same epitope, an overlapping epitope or to adjacent epitopes (e.g.,as evidenced by steric hindrance).

Other competitive binding assays include: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al. (1983)Methods in Enzymology 9:242); solid phase direct biotin-avidin EIA (seeKirkland et al. (1986) J. Immunol. 137:3614); solid phase direct labeledassay, solid phase direct labeled sandwich assay (see Harlow and Lane(1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Press);solid phase direct label RIA using I-125 label (see Morel et al. (1988)Mol. Immunol. 25(1):7); solid phase direct biotin-avidin EIA (Cheung etal. (1990) Virology 176:546); and direct labeled RIA. (Moldenhauer etal. (1990) Scand. J. Immunol. 32:77).

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen but not to other antigens.Typically, the antibody (i) binds with an equilibrium dissociationconstant (K_(D)) of approximately less than 10⁻⁷ M, such asapproximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by, e.g., surface plasmon resonance (SPR) technology in aBIACORE 2000 surface plasmon resonance instrument using thepredetermined antigen, e.g., recombinant human CD40, as the analyte andthe antibody as the ligand, or Scatchard analysis of binding of theantibody to antigen positive cells, and (ii) binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.Accordingly, an antibody that “specifically binds to human CD40” refersto an antibody that binds to soluble or cell bound human CD40 with aK_(D) of 10⁻⁷ M or less, such as approximately less than 10⁻⁸ M, 10⁻⁹ Mor 10⁻¹⁰ M or even lower. An antibody that “cross-reacts with cynomolgusCD40” refers to an antibody that binds to cynomolgus CD40 with a K_(D)of 10⁻⁷ M or less, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower.

The term “k_(assoc)” or “K_(A)”, as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction,whereas the term “k_(dis)” or “K_(D),” as used herein, refers to thedissociation rate constant of a particular antibody-antigen interaction.The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant, which is obtained from the ratio of K_(D) to K_(A) (i.e.,K_(D)/K_(A)) and is expressed as a molar concentration (M). K_(D) valuesfor antibodies can be determined using methods well established in theart. A preferred method for determining the K_(D) of an antibody isbiolayer interferometry (BLI) analysis, preferably using a ForteBioOctet RED device, surface plasmon resonance, preferably using abiosensor system such as a BIACORE surface plasmon resonance system (seeExample 5), or flow cytometry and Scatchard analysis.

The term “EC50” in the context of an in vitro or in vivo assay using anantibody or antigen binding fragment thereof, refers to theconcentration of an antibody or an antigen-binding fragment thereof thatinduces a response that is 50% of the maximal response, i.e., halfwaybetween the maximal response and the baseline.

The term “binds to immobilized CD40” refers to the ability of anantibody described herein to bind to CD40, for example, expressed on thesurface of a cell or attached to a solid support.

The term “cross-reacts,” as used herein, refers to the ability of anantibody described herein to bind to CD40 from a different species. Forexample, an antibody described herein that binds human CD40 may alsobind CD40 from another species (e.g., cynomolgus CD40). As used herein,cross-reactivity may be measured by detecting a specific reactivity withpurified antigen in binding assays (e.g., SPR, ELISA) or binding to, orotherwise functionally interacting with, cells physiologicallyexpressing CD40. Methods for determining cross-reactivity includestandard binding assays as described herein, for example, by BIACOREsurface plasmon resonance (SPR) analysis using a BIACORE 2000 SPRinstrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutivelylinked amino acid residues, with no upper limit on the length of thechain. One or more amino acid residues in the protein may contain amodification such as, but not limited to, glycosylation, phosphorylationor a disulfide bond. A “protein” may comprise one or more polypeptides.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, and may be cDNA.

Also provided are “conservative sequence modifications” to the antibodysequence provided herein, i.e. nucleotide and amino acid sequencemodifications that do not abrogate the binding of the antibody encodedby the nucleotide sequence or containing the amino acid sequence, to theantigen. For example, modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis.

Conservative sequence modifications include conservative amino acidsubstitutions, in which the amino acid residue is replaced with an aminoacid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in an anti-CD40 antibody is preferably replaced withanother amino acid residue from the same side chain family. Methods ofidentifying nucleotide and amino acid conservative substitutions that donot eliminate antigen binding are well-known in the art. See, e.g.,Brummell et al. (1993) Biochem. 32:1180-1187; Kobayashi et al. (1999)Protein Eng. 12(10):879-884; and Burks et al. (1997) Proc. Natl. Acad.Sci. (USA) 94:412-417.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of an anti-CD40 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-CD40antibodies can be screened for improved binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

For polypeptides, the term “substantial homology” indicates that twopolypeptides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate amino acid insertions ordeletions, in at least about 80% of the amino acids, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of theamino acids.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences when the sequences areoptimally aligned (i.e., % homology=# of identical positions/total # ofpositions×100), with optimal alignment determined taking into accountthe number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weightof 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide oramino acid sequences can also be determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needleman and Wunsch ((1970) J. Mol. Biol.(48):444-453) algorithm which has been incorporated into the GAP programin the GCG software package, using either a Blossum 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform a search against public databasesto, for example, identify related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules described herein. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids (e.g., the other parts of the chromosome) or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, also included are other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that comprises a nucleic acidthat is not naturally present in the cell, and may be a cell into whicha recombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

An “immune response” refers to a biological response within a vertebrateagainst foreign agents, which response protects the organism againstthese agents and diseases caused by them. An immune response is mediatedby the action of a cell of the immune system (for example, a Tlymphocyte, B lymphocyte, natural killer (NK) cell, macrophage,eosinophil, mast cell, dendritic cell or neutrophil) and solublemacromolecules produced by any of these cells or the liver (includingantibodies, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom the vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues. An immune reaction includes, e.g., activation or inhibition ofa T cell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+T cell, or the inhibition or depletion of a T_(seg) cell. “T effector”(“T_(eff)”) cells refers to T cells (e.g., CD4+ and CD8+ T cells) withcytolytic activities as well as T helper (Th) cells, which secretecytokines and activate and direct other immune cells, but does notinclude regulatory T cells (T_(reg) cells).

As used herein, the term “T cell-mediated response” refers to a responsemediated by T cells, including effector T cells (e.g., CD8+ cells) andhelper T cells (e.g., CD4+ cells). T cell mediated responses include,for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8+ T cells.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., acomponent of a signaling pathway that may be involved in modulating,regulating, or modifying an immune response. “Modulating,” “regulating,”or “modifying” an immune response refers to any alteration in a cell ofthe immune system or in the activity of such cell (e.g., an effector Tcell). Such modulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunomodulators have been identified,some of which may have enhanced function in a tumor microenvironment. Inpreferred embodiments, the immunomodulator is located on the surface ofa T cell. An “immunomodulatory target” or “immunoregulatory target” isan immunomodulator that is targeted for binding by, and whose activityis altered by the binding of, a substance, agent, moiety, compound ormolecule. Immunomodulatory targets include, for example, receptors onthe surface of a cell (“immunomodulatory receptors”) and receptorligands (“immunomodulatory ligands”).

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Immunostimulating therapy” or “immunostimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency may be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (e.g., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, “administering” refers to the physical introduction of acomposition comprising a therapeutic agent to a subject, using any ofthe various methods and delivery systems known to those skilled in theart. Preferred routes of administration for antibodies described hereininclude intravenous, intraperitoneal, intramuscular, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal,intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, the terms “inhibits” or “blocks” are usedinterchangeably and encompass both partial and completeinhibition/blocking by at least about 50%, for example, at least about60%, 70%, 80%, 90%, 95%, 99%, or 100%.

As used herein, “cancer” refers a broad group of diseases characterizedby the uncontrolled growth of abnormal cells in the body. Unregulatedcell division may result in the formation of malignant tumors or cellsthat invade neighboring tissues and may metastasize to distant parts ofthe body through the lymphatic system or bloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent to, the subject with the objective of reversing,alleviating, ameliorating, inhibiting, or slowing down or preventing theprogression, development, severity or recurrence of a symptom,complication, condition or biochemical indicia associated with adisease. Prophylaxis refers to administration to a subject who does nothave a disease, to prevent the disease from occurring or minimize itseffects if it does.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve a desired effect. A“therapeutically effective amount” or “therapeutically effective dosage”of a drug or therapeutic agent is any amount of the drug that, when usedalone or in combination with another therapeutic agent, promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction. A“prophylactically effective amount” or a “prophylactically effectivedosage” of a drug is an amount of the drug that, when administered aloneor in combination with another therapeutic agent to a subject at risk ofdeveloping a disease or of suffering a recurrence of disease, inhibitsthe development or recurrence of the disease. The ability of atherapeutic or prophylactic agent to promote disease regression orinhibit the development or recurrence of the disease can be evaluatedusing a variety of methods known to the skilled practitioner, such as inhuman subjects during clinical trials, in animal model systemspredictive of efficacy in humans, or by assaying the activity of theagent in in vitro assays.

By way of example, an anti-cancer agent is a drug that slows cancerprogression or promotes cancer regression in a subject. In preferredembodiments, a therapeutically effective amount of the drug promotescancer regression to the point of eliminating the cancer. “Promotingcancer regression” means that administering an effective amount of thedrug, alone or in combination with an anti-neoplastic agent, results ina reduction in tumor growth or size, necrosis of the tumor, a decreasein severity of at least one disease symptom, an increase in frequencyand duration of disease symptom-free periods, a prevention of impairmentor disability due to the disease affliction, or otherwise ameliorationof disease symptoms in the patient. Pharmacological effectiveness refersto the ability of the drug to promote cancer regression in the patient.Physiological safety refers to an acceptably low level of toxicity, orother adverse physiological effects at the cellular, organ and/ororganism level (adverse effects) resulting from administration of thedrug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount or dosage of the drug preferably inhibits cell growthor tumor growth by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. In themost preferred embodiments, a therapeutically effective amount or dosageof the drug completely inhibits cell growth or tumor growth, i.e.,preferably inhibits cell growth or tumor growth by 100%. The ability ofa compound to inhibit tumor growth can be evaluated using the assaysdescribed infra. Inhibition of tumor growth may not be immediate aftertreatment, and may only occur after a period of time or after repeatedadministration. Alternatively, this property of a composition can beevaluated by examining the ability of the compound to inhibit cellgrowth, such inhibition can be measured in vitro by assays known to theskilled practitioner. In other preferred embodiments described herein,tumor regression may be observed and may continue for a period of atleast about 20 days, more preferably at least about 40 days, or evenmore preferably at least about 60 days.

“Combination” therapy, as used herein, unless otherwise clear from thecontext, is meant to encompass administration of two or more therapeuticagents in a coordinated fashion, and includes, but is not limited to,concurrent dosing. Specifically, combination therapy encompasses bothco-administration (e.g. administration of a co-formulation orsimultaneous administration of separate therapeutic compositions) andserial or sequential administration, provided that administration of onetherapeutic agent is conditioned in some way on administration ofanother therapeutic agent. For example, one therapeutic agent may beadministered only after a different therapeutic agent has beenadministered and allowed to act for a prescribed period of time. See,e.g., Kohrt et al. (2011) Blood 117:2423.

The terms “patient” and “subject” refer to any human that receiveseither prophylactic or therapeutic treatment. For example, the methodsand compositions described herein can be used to treat a subject havingcancer.

Various aspects described herein are described in further detail in thefollowing subsections.

I. Anti-CD40 Antibodies

The present application discloses agonistic anti-huCD40 antibodieshaving desirable properties for use as therapeutic agents in treatingdiseases such as cancers. These properties include one or more of theability to bind to human CD40 with high affinity, acceptably lowimmunogenicity in human subjects, the ability to bind preferentially toFcγRIIb, and the absence of sequence liabilities that might reduce thechemical stability of the antibody.

The anti-CD40 antibodies disclosed herein by sequence bind to specificepitopes on human CD40. Other antibodies that bind to the same orclosely related epitopes would likely share these desirable properties,and may be discovered doing competition experiments.

Anti-huCD40 Antibodies that Compete with Anti-huCD40 AntibodiesDisclosed Herein

Anti-huCD40 antibodies that compete with the antibodies of the presentinvention for binding to huCD40 may be raised using immunizationprotocols similar to those described herein (Examples 1 and 2).Antibodies that compete for binding with the anti-huCD40 antibodiesdisclosed herein by sequence may also be generated by immunizing mice orother non-human animal with human CD40 or a construct comprising theextracellular domain thereof (residues 21-193 of SEQ ID NO: 11), or byimmunizing with a fragment of human CD40 containing the epitope bound bythe anti-huCD40 antibodies disclosed herein. The resulting antibodiescan be screened for the ability to block binding of an antibodycomprising a mutant Fc region having one or more mutations correspondingto one or more mutations in an IgG havey chain selected from the groupconsisting of N297A, SE, SELF, V9, and or V11 (SEQ ID Nos: 3-7), tohuman CD40 by methods well known in the art, for example blockingbinding to fusion protein of the extracellular domain of CD40 and animmunoglobulin Fc domain in a ELISA, or blocking the ability to bind tocells expressing huCD40 on their surface, e.g. by FACS. In variousembodiments, the test antibody is contacted with the CD40-Fc fusionprotein (or to cells expressing huCD40 on their surface) prior to, atthe same time as, or after the addition of an antibody comprising amutant Fc region having one or more mutations corresponding to one ormore mutations in an IgG havey chain selected from the group consistingof N297A, SE, SELF, V9, and or V11 (SEQ ID Nos: 3-7). For example,“binning” experiments may be performed to determine whether a testantibody falls into the same “bin” as an antibodies disclosed herein bysequence, with antibodies disclosed herein by sequence as the“reference” antibodies and the antibodies to be tested as the “test”antibodies. Antibodies that reduce binding of the antibodies disclosedherein by sequence to human CD40 (either as an Fc fusion or on a cell),particularly at roughly stoichiometric concentrations, are likely tobind at the same, overlapping, or adjacent epitopes, and thus may sharethe desirable functional properties of an antibody comprising a mutantFc region having one or more mutations corresponding to one or moremutations in an IgG havey chain selected from the group consisting ofN297A, SE, SELF, V9, and or V11 (SEQ ID Nos: 3-7).

Accordingly, provided herein are anti-huCD40 antibodies that inhibit thebinding of an anti-huCD40 antibodies described herein to huCD40 on cellsby at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or by 100%, and/orwhose binding to huCD40 on cells is inhibited by an anti-huCD40antibodies described herein by at least 10%, 20%, 30%, 40%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or by 100%, e.g., as measured by ELISA or FACS, such as byusing the assay described in the following paragraph.

An exemplary competition experiment to determine whether a test antibodyblocks the binding of (i.e., “competes with”) a reference antibody, maybe conducted as follows: cells expressing CD40 are seeded at 105 cellsper sample well in a 96 well plate. The plate is set on ice followed bythe addition of unconjugated test antibody at concentrations rangingfrom 0 to 50 μg/mL (three-fold titration starting from a highestconcentration of 50 μg/mL). An unrelated IgG may be used as an isotypecontrol for the first antibody and added at the same concentrations(three-fold titration starting from a highest concentration of 50μg/mL). A sample pre-incubated with 50 μg/mL unlabeled referenceantibody may be included as a positive control for complete blocking(100% inhibition) and a sample without antibody in the primaryincubation may be used as a negative control (no competition; 0%inhibition). After 30 minutes of incubation, labeled, e.g.,biotinylated, reference antibody is added at a concentration of 2 μg/mLper well without washing. Samples are incubated for another 30 minuteson ice. Unbound antibodies are removed by washing the cells with FACSbuffer. Cell-bound labeled reference antibody is detected with an agentthat detects the label, e.g., PE conjugated streptavidin (Invitrogen,catalog #S21388) for detecting biotin. The samples are acquired on aFACS Calibur Flow Cytometer (BD, San Jose) and analyzed with FLOWJOsoftware (Tree Star, Inc, Ashland, OR). The results may be representedas the % inhibition (i.e., subtracting from 100% the amount of label ateach concentration divided by the amount of label obtained with noblocking antibody).

Typically, the same experiment is then conducted in the reverse, i.e.,the test antibody is the reference antibody and the reference antibodyis the test antibody. In certain embodiments, an antibody at leastpartially (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90%) or completely (100%) blocks the binding of the other antibody tothe target, e.g. human CD40 or fragment thereof, and regardless ofwhether inhibition occurs when one or the other antibody is thereference antibody. A reference antibody and a test antibody“cross-block” binding of each other to the target when the antibodiescompete with each other both ways, i.e., in competition experiments inwhich the reference antibody is added first and in competitionexperiments in which the test antibody is added first.

Anti-huCD40 Antibodies that Bind to the Same Epitope

Anti-huCD40 antibodies that bind to the same or similar epitopes to theantibodies disclosed herein may be raised using immunization protocolssimilar to those described herein. The resulting antibodies can bescreened for high affinity binding to human CD40. Selected antibodiescan then be studied in yeast display assay in which sequence variants ofhuCD40 are presented on the surface of yeast cells, or byhydrogen-deuterium exchange experiments, to determine the preciseepitope bound by the antibody.

Epitope determinations may be made by any method known in the art. Invarious embodiments, anti-huCD40 antibodies are considered to bind tothe same epitope as an anti-huCD40 mAb disclosed herein if they makecontact with one or more of the same residues within at least one regionof huCD40; if they make contacts with a majority of the residues withinat least one region of huCD40; if they make contacts with a majority ofthe residues within each region of huCD40; if they make contact with amajority of contacts along the entire length of huCD40; if they makecontacts within all of the same distinct regions of human CD40; if theymake contact with all of the residues at any one region on human CD40;or if they make contact with all of the same residues at all of the sameregions. Epitope “regions” are clusters of residues along the primarysequence.

Techniques for determining antibodies that bind to the “same epitope onhuCD40” with the antibodies described herein include x-ray analyses ofcrystals of antigen:antibody complexes, which provides atomic resolutionof the epitope. Other methods monitor the binding of the antibody toantigen fragments or mutated variations of the antigen where loss ofbinding due to a modification of an amino acid residue within theantigen sequence is often considered an indication of an epitopecomponent. Methods may also rely on the ability of an antibody ofinterest to affinity isolate specific short peptides (either in nativethree dimensional form or in denatured form) from combinatorial phagedisplay peptide libraries or from a protease digest of the targetprotein. The peptides are then regarded as leads for the definition ofthe epitope corresponding to the antibody used to screen the peptidelibrary. For epitope mapping, computational algorithms have also beendeveloped that have been shown to map conformational discontinuousepitopes.

The epitope or region comprising the epitope can also be identified byscreening for binding to a series of overlapping peptides spanning CD40.Alternatively, the method of Jespers et al. (1994) Biotechnology 12:899may be used to guide the selection of antibodies having the same epitopeand therefore similar properties to the an anti-CD40 antibodiesdescribed herein. Using phage display, first the heavy chain of theanti-CD40 antibody is paired with a repertoire of (preferably human)light chains to select a CD40-binding antibody, and then the new lightchain is paired with a repertoire of (preferably human) heavy chains toselect a (preferably human) CD40-binding antibody having the sameepitope or epitope region as an anti-huCD40 antibody described herein.Alternatively variants of an antibody described herein can be obtainedby mutagenesis of cDNA encoding the heavy and light chains of theantibody.

Alanine scanning mutagenesis, as described by Cunningham & Wells (1989)Science 244: 1081, or some other form of point mutagenesis of amino acidresidues in CD40 (such as the yeast display method provided at Example6) may also be used to determine the functional epitope for an anti-CD40antibody.

The epitope or epitope region (an “epitope region” is a regioncomprising the epitope or overlapping with the epitope) bound by aspecific antibody may also be determined by assessing binding of theantibody to peptides comprising fragments of CD40. A series ofoverlapping peptides encompassing the sequence of CD40 (e.g., humanCD40) may be synthesized and screened for binding, e.g. in a directELISA, a competitive ELISA (where the peptide is assessed for itsability to prevent binding of an antibody to CD40 bound to a well of amicrotiter plate), or on a chip. Such peptide screening methods may notbe capable of detecting some discontinuous functional epitopes, i.e.functional epitopes that involve amino acid residues that are notcontiguous along the primary sequence of the CD40 polypeptide chain.

An epitope may also be identified by MS-based protein footprinting, suchas hydrogen/deuterium exchange mass spectrometry (HDX-MS) and FastPhotochemical Oxidation of Proteins (FPOP). HDX-MS may be conducted,e.g., as further described at Wei et al. (2014) Drug Discovery Today19:95, the methods of which are specifically incorporated by referenceherein. FPOP may be conducted as described, e.g., in Hambley & Gross(2005) J. American Soc. Mass Spectrometry 16:2057, the methods of whichare specifically incorporated by reference herein.

The epitope bound by anti-CD40 antibodies may also be determined bystructural methods, such as X-ray crystal structure determination (e.g.,WO 2005/044853), molecular modeling and nuclear magnetic resonance (NMR)spectroscopy, including NMR determination of the H-D exchange rates oflabile amide hydrogens in CD40 when free and when bound in a complexwith an antibody of interest (Zinn-Justin et al. (1992)Biochemistry31:11335; Zinn-Justin et al. (1993)Biochemistry 32:6884).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339; McPherson (1990) Eur. J. Biochem.189:1), including microbatch (e.g. Chayen (1997) Structure 5:1269),hanging-drop vapor diffusion (e.g. McPherson (1976) J. Biol. Chem.251:6300), seeding and dialysis. It is desirable to use a proteinpreparation having a concentration of at least about 1 mg/mL andpreferably about 10 mg/mL to about 20 mg/mL. Crystallization may be bestachieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst.D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Unless otherwise indicated, and with reference to the claims, theepitope bound by an antibody is the epitope as determined by HDX-MSmethods.

Anti-CD40 Antibodies that Bind with High Affinity

In some embodiments the anti-huCD40 antibodies of the present inventionbind to huCD40 with high affinity, like the anti-huCD40 antibodiesdisclosed herein, increasing their likelihood of being effectivetherapeutic agents. In various embodiments anti-huCD40 antibodies of thepresent invention bind to huCD40 with a K_(D) of less than 10 nM, 5 nM,2 nM, 1 nM, 300 pM or 100 pM. In other embodiments, the anti-huCD40antibodies of the present invention bind to huCD40 with a K_(D) between2 nM and 100 pM. Standard assays to evaluate the binding ability of theantibodies toward huCD40 include ELISAs, RIAs, Western blots, biolayerinterferometry (BLI) and BIACORE SPR analysis.

Anti-CD40 Antibody Sequence Variants

Some variability in the antibody sequences disclosed herein may betolerated and still maintain the desirable properties of the antibody.The CDR regions are delineated using the Kabat system (Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242). Accordingly, the present invention further providesanti-huCD40 antibodies comprising CDR sequences that are at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the CDRsequences of the antibodies disclosed herein. The present invention alsoprovides anti-huCD40 antibodies comprising heavy and/or light chainvariable domain sequences that are at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the heavy and/or light chainvariable domain sequences of an antibody comprising a mutant Fc regionhaving one or more mutations corresponding to one or more mutations inan IgG havey chain selected from the group consisting of N297A, SE,SELF, V9, and or V11 (SEQ ID Nos: 3-7), and humanized derivativesthereof).

As used herein, a murine antibody comprises heavy or light chainvariable regions that are “derived from” a particular germline sequenceif the variable regions of the antibody are obtained from a system thatuses murine germline immunoglobulin genes, and the antibody sequence issufficiently related to the germline that it is more likely derived fromthe given germline than from any other. Such systems include immunizinga mouse with the antigen of interest. The murine germline immunoglobulinsequence(s) from which the sequence of an antibody is “derived” can beidentified by comparing the amino acid sequence of the antibody to theamino acid sequences of murine germline immunoglobulins and selectingthe germline immunoglobulin sequence that is closest in sequence (i.e.,greatest % identity) to the sequence of the antibody. A murine antibodythat is “derived from” a particular germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence dueto, for example, naturally-occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected murineantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a germline immunoglobulin gene (e.g. Vregions). In certain cases, a murine antibody may be at least 95%, oreven at least 96%, 97%, 98%, or 99% identical in amino acid sequence tothe amino acid sequence encoded by the germline immunoglobulin gene(e.g. V regions). Typically, an antibody derived from a particularmurine germline sequence will display no more than 10 amino aciddifferences from the amino acid sequence encoded by the germlineimmunoglobulin gene (e.g. V regions). In certain cases, the murineantibody may comprise no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene (e.g. V regions).

II. Engineered and Modified Antibodies

V_(H) and V_(L) Regions

Also provided are engineered and modified antibodies that can beprepared using an antibody having one or more of the V_(H) and/or V_(L)sequences disclosed herein as starting material to engineer a modifiedantibody, which modified antibody may have altered properties from thestarting antibody. An antibody can be engineered by modifying one ormore residues within one or both variable regions (i.e., V_(H) and/orV_(L)), for example within one or more CDR regions and/or within one ormore framework regions. Additionally or alternatively, an antibody canbe engineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Such grafting is of particular use in humanizing non-humananti-CD40 antibodies that compete for binding with the anti-huCD40antibodies disclosed herein and/or bind to the same epitope as theanti-huCD40 antibodies disclosed herein. Antibodies interact with targetantigens predominantly through amino acid residues that are located inthe six heavy and light chain complementarity determining regions(CDRs). For this reason, the amino acid sequences within CDRs are morediverse between individual antibodies than sequences outside of CDRs.Because CDR sequences are responsible for most antibody-antigeninteractions, it is possible to express recombinant antibodies thatmimic the properties of specific reference antibodies by constructingexpression vectors that include CDR sequences from the specificreference antibody grafted onto framework sequences from a differentantibody with different properties (see, e.g., Riechmann, L. et al.(1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525;Queen, C. et al. (1989) Proc. Natl. Acad. See. (USA) 86:10029-10033;U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101;5,585,089; 5,693,762 and 6,180,370 to Queen et al.)

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase, as well as in Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) “The Repertoire of Human Germline V_(H) SequencesReveals about Fifty Groups of V_(H) Segments with DifferentHypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al.(1994) “A Directory of Human Germ-line V_(H) Segments Reveals a StrongBias in their Usage” Eur. J. Immunol. 24:827-836; the contents of eachof which are expressly incorporated herein by reference.

Preferred framework sequences for use in the antibodies described hereinare those that are structurally similar to the framework sequences usedby antibodies described herein. The V_(H) CDR1, 2 and 3 sequences, andthe V_(L) CDR1, 2 and 3 sequences, can be grafted onto framework regionsthat have the identical sequence as that found in the germlineimmunoglobulin gene from which the framework sequence derive, or the CDRsequences can be grafted onto framework regions that contain up to 20,preferably conservative, amino acid substitutions as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen el al.).

Engineered antibodies described herein include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Often suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to be encompassed.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

Another type of variable region modification is to mutate amino acidresidues within the CDR regions to improve one or more bindingproperties (e.g., affinity) of the antibody of interest. Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest. Preferably conservative modifications areintroduced. The mutations may be amino acid additions, deletions, orpreferably substitutions. Moreover, typically no more than one, two,three, four or five residues within a CDR region are altered.

Methionine residues in CDRs of antibodies can be oxidized, resulting inpotential chemical degradation and consequent reduction in potency ofthe antibody. Accordingly, also provided are anti-CD40 antibodies thathave one or more methionine residues in the heavy and/or light chainCDRs replaced with amino acid residues that do not undergo oxidativedegradation. Similarly, deamidation sites may be removed from anti-CD40antibodies, particularly in the CDRs. Potential glycosylation siteswithin the antigen binding domain are preferably eliminated to preventglycosylation that may interfere with antigen binding. See, e.g., U.S.Pat. No. 5,714,350.

Fcs and Modified Fcs

Antibodies of the present invention may comprise the variable domains ofthe invention combined with constant domains comprising different Fcregions, selected based on the biological activities (if any) of theantibody for the intended use. Salfeld (2007) Nat. Biotechnol. 25:1369.Human IgGs, for example, can be classified into four subclasses, IgG1,IgG2, IgG3, and IgG4, and each these of these comprises an Fc regionhaving a unique profile for binding to one or more of Fcγ receptors(activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32a,c); FcγRIIIAand FcγRIIIB (CD16a,b) and inhibiting receptor FcγRIIB (CD32b), and forthe first component of complement (C1q). Human IgG1 and IgG3 bind to allFcγ receptors; IgG2 binds to FcγRIIAH131, and with lower affinity toFcγRIIAR131 FcγRIIIAV158; IgG4 binds to FcγRI, FcγRIIA, FcγRIIB,FcγRIIC, and FcγRIIIAV158; and the inhibitory receptor FcγRIIB has alower affinity for IgG1, IgG2 and IgG3 than all other Fcγ receptors.Bruhns et al. (2009) Blood 113:3716. Studies have shown that FcγRI doesnot bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Id. Ingeneral, with regard to ADCC activity, human IgG1≥IgG3≥≥IgG4≥IgG2. As aconsequence, for example, an IgG1 constant domain, rather than an IgG2or IgG4, might be chosen for use in a drug where ADCC is desired; IgG3might be chosen if activation of FcγRIIIA-expressing NK cells, monocytesof macrophages; and IgG4 might be chosen if the antibody is to be usedto desensitize allergy patients. IgG4 may also be selected if it isdesired that the antibody lack all effector function.

Anti-huCD40 variable regions described herein may be linked (e.g.,covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3 or IgG4Fc, which may be of any allotype or isoallotype, e.g., for IgG1: G1m,G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3:G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4),G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v).See, e.g., Jefferis et al. (2009) mAbs 1:1). Selection of allotype maybe influenced by the potential immunogenicity concerns, e.g. to minimizethe formation of anti-drug antibodies.

In preferred embodiments, anti-CD40 antibodies of the present inventionhave an Fc that binds to or has enhanced binding to FcγRIIb, which canprovide enhanced agonism. See, e.g., WO 2012/087928; Li & Ravetch (2011)Science 333:1030; Wilson et al. (2011) Cancer Cell 19:101; White et al.(2011) J. Immunol. 187:1754. Variable regions described herein may belinked to Fc variants that enhance affinity for the inhibitory receptorFcγRIIb, e.g. to enhance apoptosis-inducing or adjuvant activity. Li &Ravetch (2012) Proc. Nat'l Acad. Sci. (USA) 109:10966; U.S. PatentApplication Publication No. 2014/0010812. Such variants may provide anantibody with immunomodulatory activities related to FcγRIIb+ cells,including for example B cells and monocytes. In one embodiment, the Fcvariants provide selectively enhanced affinity to FcγRIIb relative toone or more activating receptors. Such variants may also exhibitenhanced FcR-mediated cross-linking, resulting in enhanced therapeuticefficacy. Modifications for altering binding to FcγRIIb include one ormore modifications at a position selected from the group consisting of234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332,according to the EU index. Exemplary substitutions for enhancing FcγRIIbaffinity include but are not limited to 234D, 234E, 234F, 234W, 235D,235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E,268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplarysubstitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F,328W, and 328Y. Other Fc variants for enhancing binding to FcγRIIbinclude 235Y-267E, 236D-267E, 239D-268D, 239D-267E, 267E-268D,267E-268E, and 267E-328F. Specifically, the S267E, G236D, S239D, L328Fand 1332E variants, including the S267E-L328F double variant, of humanIgG1 are of particular value in specifically enhancing affinity for theinhibitory FcγRIIb receptor. Chu et al. (2008)Mol. Immunol. 45:3926;U.S. Patent Application Publication No. 2006/024298; WO 2012/087928.Enhanced specificity for FcγRIIb (as distinguished from FcγRIIa_(R131))may be obtained by adding the P238D substitution and other mutations(Mimoto et al. (2013) Protein. Eng. Des. & Selection 26:589; WO2012/1152410), as well as V262E and V264E (Yu et al. (2013) J. Am. Chem.Soc. 135:9723, and WO 2014/184545. See Table 2.

Half-life Extension

In certain embodiments, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, thismay be done by increasing the binding affinity of the Fc region forFcRn. In one embodiment, the antibody is altered within the CH1 or CLregion to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary Fcvariants that increase binding to FcRn and/or improve pharmacokineticproperties include substitutions at positions 259, 308, and 434,including for example 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y,and 434M. Other variants that increase Fc binding to FcRn include: 250E,250Q, 428L, 428F, 250Q/428L (Hinton et al., (2004), J. Biol. Chem.279(8): 6213-6216, Hinton et al. (2006) Journal of Immunology176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A(Shields et al., (2001) Journal of Biological Chemistry,276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F,434Y, 252Y/254T/256E, 433K/434F/436H (Dall'Acqua et al. (2002) Journalof Immunology, 169:5171-5180, Dall'Acqua et al., (2006), Journal ofBiological Chemistry 281:23514-23524). See U.S. Pat. No. 8,367,805.

Modification of certain conserved residues in IgG Fc (1253, H310, Q311,H433, N434), such as the N434A variant (Yeung et al. (2009) J. Immunol.182:7663), have been proposed as a way to increase FcRn affinity, thusincreasing the half-life of the antibody in circulation. WO 98/023289.The combination Fc variant comprising M428L and N434S has been shown toincrease FcRn binding and increase serum half-life up to five-fold.Zalevsky et al. (2010) Nat. Biotechnol. 28:157. The combination Fcvariant comprising T307A, E380A and N434A modifications also extendshalf-life of IgG1 antibodies. Petkova et al. (2006) Int. Immunol.18:1759. In addition, combination Fc variants comprising M252Y-M428L,M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, andM428L-N434S variants have also been shown to extend half-life. WO2009/086320.

Further, a combination Fc variant comprising M252Y, S254T and T256E,increases half-life-nearly 4-fold. Dall'Acqua et al. (2006) J. Biol.Chem. 281:23514. A related IgG1 modification providing increased FcRnaffinity but reduced pH dependence (M252Y-S254T-T256E-H433K-N434F) hasbeen used to create an IgG1 construct (“MST-HN Abdeg”) for use as acompetitor to prevent binding of other antibodies to FcRn, resulting inincreased clearance of that other antibody, either endogenous IgG (e.g.in an autoimmune setting) or another exogenous (therapeutic) mAb.Vaccaro et al. (2005) Nat. Biotechnol. 23:1283; WO 2006/130834.

Other modifications for increasing FcRn binding are described in Yeunget al. (2010) J. Immunol. 182:7663-7671; 6,277,375; 6,821,505; WO97/34631; WO 2002/060919.

In certain embodiments, hybrid IgG isotypes may be used to increase FcRnbinding, and potentially increase half-life. For example, an IgG1/IgG3hybrid variant may be constructed by substituting IgG1 positions in theCH2 and/or CH3 region with the amino acids from IgG3 at positions wherethe two isotypes differ. Thus a hybrid variant IgG antibody may beconstructed that comprises one or more substitutions, e.g., 274Q, 276K,300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In otherembodiments described herein, an IgG1/IgG2 hybrid variant may beconstructed by substituting IgG2 positions in the CH2 and/or CH3 regionwith amino acids from IgG1 at positions where the two isotypes differ.Thus a hybrid variant IgG antibody may be constructed that comprises oneor more substitutions, e.g., one or more of the following amino acidsubstitutions: 233E, 234L, 235L, −236G (referring to an insertion of aglycine at position 236), and 327A. See U.S. Pat. No. 8,629,113. Ahybrid of IgG1/IgG2/IgG4 sequences has been generated that purportedlyincreases serum half-life and improves expression. U.S. Pat. No.7,867,491 (sequence number 18 therein).

The serum half-life of the antibodies of the present invention can alsobe increased by pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with a polyethylene glycol (PEG) reagent, such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. Preferably, the pegylation is carried out via an acylationreaction or an alkylation reaction with a reactive PEG molecule (or ananalogous reactive water-soluble polymer). As used herein, the term“polyethylene glycol” is intended to encompass any of the forms of PEGthat have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.In certain embodiments, the antibody to be pegylated is an aglycosylatedantibody. Methods for pegylating proteins are known in the art and canbe applied to the antibodies described herein. See for example, EP0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

Alternatively, under some circumstances it may be desirable to decreasethe half-life of an antibody of the present invention, rather thanincrease it. Modifications such as I253A (Hornick et al. (2000) J. Nucl.Med. 41:355) and H435A/R, I253A or H310A (Kim et al. (2000) Eur. J.Immunol. 29:2819) in Fc of human IgG1 can decrease FcRn binding, thusdecreasing half-life (increasing clearance) for use in situations whererapid clearance is preferred, such a medical imaging. See also Kenanovaet al. (2005) Cancer Res. 65:622. Other means to enhance clearanceinclude formatting the antigen binding domains of the present inventionas antibody fragments lacking the ability to bind FcRn, such as Fabfragments. Such modification can reduce the circulating half-life of anantibody from a couple of weeks to a matter of hours. SelectivePEGylation of antibody fragments can then be used to fine-tune(increase) the half-life of the antibody fragments if necessary. Chapmanet al. (1999)Nat. Biotechnol. 17:780. Antibody fragments may also befused to human serum albumin, e.g. in a fusion protein construct, toincrease half-life. Yeh et al. (1992) Proc. Nat'l Acad. Sci. (USA)89:1904. Alternatively, a bispecific antibody may be constructed with afirst antigen binding domain of the present invention and a secondantigen binding domain that binds to human serum albumin (HSA). SeeInt'l Pat. Appl. Pub. WO 2009/127691 and patent references citedtherein. Alternatively, specialized polypeptide sequences can be addedto antibody fragments to increase half-life, e.g. “XTEN” polypeptidesequences. Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; Int'lPat. Appl. Pub. WO 2010/091122.

Additional Fc Variants

When using an IgG4 constant domain, it is usually preferable to includethe substitution S228P, which mimics the hinge sequence in IgG1 andthereby stabilizes IgG4 molecules, e.g. reducing Fab-arm exchangebetween the therapeutic antibody and endogenous IgG4 in the patientbeing treated. Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy etal. (2000) J. Immunol. 164:1925.

A potential protease cleavage site in the hinge of IgG1 constructs canbe eliminated by D221G and K222S modifications, increasing the stabilityof the antibody. WO 2014/043344.

The affinities and binding properties of an Fc variant for its ligands(Fc receptors) may be determined by a variety of in vitro assay methods(biochemical or immunological based assays) known in the art includingbut not limited to, equilibrium methods (e.g., enzyme-linkedimmunosorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics(e.g., BIACORE SPR analysis), and other methods such as indirect bindingassays, competitive inhibition assays, fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

In still other embodiments, the glycosylation of an antibody is modifiedto increase or decrease effector function. For example, an aglycoslatedantibody can be made that lacks all effector function by mutating theconserved asparagine residue at position 297 (e.g. N297A), thusabolishing complement and FcγRI binding. Bolt et al. (1993) Eur. J.Immunol. 23:403. See also Tao & Morrison (1989) J. Immunol. 143:2595(using N297Q in IgG1 to eliminate glycosylation at position 297).

Although aglycosylated antibodies generally lack effector function,mutations can be introduced to restore that function. Aglycosylatedantibodies, e.g. those resulting from N297A/C/D/or H mutations orproduced in systems (e.g. E. coli) that do not glycosylate proteins, canbe further mutated to restore FcγR binding, e.g. S298G and/or T299A/G/orH (WO 2009/079242), or E382V and M428I (Jung et al. (2010) Proc. Nat'lAcad. Sci. (USA) 107:604).

Glycoengineering can also be used to modify the anti-inflammatoryproperties of an IgG construct by changing the α2,6 sialyl content ofthe carbohydrate chains attached at Asn297 of the Fc regions, wherein anincreased proportion of α2,6 sialylated forms results in enhancedanti-inflammatory effects. See Nimmerjahn et al. (2008) Ann. Rev.Immunol. 26:513. Conversely, reduction in the proportion of antibodieshaving α2,6 sialylated carbohydrates may be useful in cases whereanti-inflammatory properties are not wanted. Methods of modifying α2,6sialylation content of antibodies, for example by selective purificationof α2,6 sialylated forms or by enzymatic modification, are provided atU.S. Patent Application Publication No. 2008/0206246. In otherembodiments, the amino acid sequence of the Fc region may be modified tomimic the effect of α2,6 sialylation, for example by inclusion of anF241A modification. WO 2013/095966.

III. Antibody Physical Properties

Antibodies described herein can contain one or more glycosylation sitesin either the light or heavy chain variable region. Such glycosylationsites may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al. (1972) Ann. Rev. Biochem. 41:673-702; Gala and Morrison(2004) J. Immunol. 172:5489-94; Wallick et al. (1988) J. Exp. Med.168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985)Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some instances, it is preferred to have an anti-huCD40antibody that does not contain variable region glycosylation. This canbe achieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

In certain embodiments, the antibodies described herein do not containasparagine isomerism sites. The deamidation of asparagine may occur onN-G or D-G sequences and result in the creation of an isoaspartic acidresidue that introduces a kink into the polypeptide chain and decreasesits stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-CD40 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

Each antibody will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr. Pharm. Biotechnol.3:361-71). Generally, it is preferred that the TM1 (the temperature ofinitial unfolding) be greater than 60° C., preferably greater than 65°C., even more preferably greater than 70° C. The melting point of anantibody can be measured using differential scanning calorimetry (Chenet al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett.68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr.Sci. 40:343-9).

In a preferred embodiment, antibodies are selected that do not degraderapidly. Degradation of an antibody can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem. 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects, which can lead to the triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation can be measured by several techniques, includingsize-exclusion column (SEC), high performance liquid chromatography(HPLC), and light scattering.

IV. Nucleic Acid Molecules

Another aspect described herein pertains to nucleic acid molecules thatencode the antibodies described herein. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids (e.g., otherchromosomal DNA, e.g., the chromosomal DNA that is linked to theisolated DNA in nature) or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, restrictionenzymes, agarose gel electrophoresis and others well known in the art.See, F. Ausubel, et al., ed. (1987) Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York. A nucleicacid described herein can be, for example, DNA or RNA and may or may notcontain intronic sequences. In a certain embodiments, the nucleic acidis a cDNA molecule.

Nucleic acids described herein can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions(hinge, CH1, CH2 and/or CH3). The sequences of human heavy chainconstant region genes are known in the art (see e.g., Kabat, E. A., elal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The heavy chain constant regioncan be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region,for example, an IgG1 region. For a Fab fragment heavy chain gene, theV_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L). The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci. (USA) 85:5879-5883; McCafferty et al., (1990)Nature348:552-554).

V. Antibody Generation

Various antibodies of the present invention, e.g. those that competewith or bind to the same epitope as the anti-human CD40 antibodiesdisclosed herein, can be produced using a variety of known techniques,such as the standard somatic cell hybridization technique described byKohler and Milstein, Nature 256: 495 (1975). Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibodies also can be employed, e.g., viral oroncogenic transformation of B lymphocytes, phage display technique usinglibraries of human antibody genes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies described herein can be prepared basedon the sequence of a murine monoclonal antibody prepared as describedabove. DNA encoding the heavy and light chain immunoglobulins can beobtained from the murine hybridoma of interest and engineered to containnon-murine (e.g., human) immunoglobulin sequences using standardmolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567 to Cabilly et al.). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

In one embodiment, the antibodies described herein are human monoclonalantibodies. Such human monoclonal antibodies directed against human CD40can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHUMAB mice and KM mice, respectively, and are collectively referred toherein as “human Ig mice.”

The HUMAB mouse (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μand γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HUMAB mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. (USA)90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In certain embodiments, antibodies described herein are raised using amouse that carries human immunoglobulin sequences on transgenes andtranschromosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-huCD40 antibodies described herein.

For example, an alternative transgenic system referred to as theXenomouse (Abgenix, Inc.) can be used; such mice are described in, forexample, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6, 150,584 and6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CD40 antibodies described herein. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. (USA)97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-huCD40antibodies described herein.

Additional mouse systems described in the art for raising humanantibodies, e.g., human anti-huCD40 antibodies, include (i) theVELOCIMMUNE mouse (Regeneron Pharmaceuticals, Inc.), in which theendogenous mouse heavy and light chain variable regions have beenreplaced, via homologous recombination, with human heavy and light chainvariable regions, operatively linked to the endogenous mouse constantregions, such that chimeric antibodies (human V/mouse C) are raised inthe mice, and then subsequently converted to fully human antibodiesusing standard recombinant DNA techniques; and (ii) the MEMO mouse(Merus Biopharmaceuticals, Inc.), in which the mouse containsunrearranged human heavy chain variable regions but a single rearrangedhuman common light chain variable region. Such mice, and use thereof toraise antibodies, are described in, for example, WO 2009/15777, US2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal antibodies described herein can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies described herein can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunizations

To generate fully human antibodies to human CD40, mice or transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the CD40 antigen and/or cells expressing CD40, asdescribed for other antigens, for example, by Lonberg et al. (1994)Nature 368(6474): 856-859; Fishwild et al. (1996) Nature Biotechnology14: 845-851 and WO 98/24884. Alternatively, mice can be immunized withDNA encoding human CD40. Preferably, the mice will be 6-16 weeks of ageupon the first infusion. For example, a purified or enriched preparation(5-50 μg) of the recombinant human CD40 antigen can be used to immunizethe mice intraperitoneally. In the event that immunizations using apurified or enriched preparation of the CD40 antigen do not result inantibodies, mice can also be immunized with cells expressing CD40, e.g.,a cell line, to promote immune responses.

Cumulative experience with various antigens has shown that the HUMABtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in Ribi's adjuvant, followed byevery other week IP/SC immunizations (up to a total of 10) with antigenin Ribi's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA and FACS (asdescribed below), and mice with sufficient titers of anti-CD40 humanimmunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen and lymph nodes. It is expected that 2-3 fusions for eachimmunization may need to be performed. Between 6 and 24 mice aretypically immunized for each antigen. Usually, HCo7, HCo12, and KMstrains are used. In addition, both HCo7 and HCo12 transgene can be bredtogether into a single mouse having two different human heavy chaintransgenes (HCo7/HCo12).

Generation of Hybridomas Producing Monoclonal Antibodies to CD40

To generate hybridomas producing monoclonal antibodies described herein,splenocytes and/or lymph node cells from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line. The resulting hybridomas can be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toSp2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG.Cells are plated at approximately 2×10′ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 10%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma). After approximately two weeks, cellscan be cultured in medium in which the HAT is replaced with HT.Individual wells can then be screened by ELISA for human monoclonal IgMand IgG antibodies. Once extensive hybridoma growth occurs, medium canbe observed usually after 10-14 days. The antibody secreting hybridomascan be replated, screened again, and if still positive for human IgG,the monoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify monoclonal antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

VI. Antibody Manufacture Generation of Transfectomas ProducingMonoclonal Antibodies to CD40

Antibodies of the present invention, including both specific antibodiesfor which sequences are provided and other, related anti-CD40antibodies, can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (Morrison, S. (1985)Science 229:1202).

For example, to express antibodies, or antibody fragments thereof, DNAsencoding partial or full-length light and heavy chains, can be obtainedby standard molecular biology techniques (e.g., PCR amplification orcDNA cloning using a hybridoma that expresses the antibody of interest)and the DNAs can be inserted into expression vectors such that the genesare operatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or both genes are insertedinto the same expression vector. The antibody genes are inserted intothe expression vector(s) by standard methods (e.g., ligation ofcomplementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present). Thelight and heavy chain variable regions of the antibodies describedherein can be used to create full-length antibody genes of any antibodyisotype by inserting them into expression vectors already encoding heavychain constant and light chain constant regions of the desired isotypesuch that the V_(H) segment is operatively linked to the CH segment(s)within the vector and the V_(L) segment is operatively linked to theC_(L) segment within the vector. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, recombinant expression vectorsmay carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel (Gene Expression Technology.Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Itwill be appreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences, maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV), SimianVirus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may beused, such as the ubiquitin promoter or β-globin promoter. Stillfurther, regulatory elements composed of sequences from differentsources, such as the SRα promoter system, which contains sequences fromthe SV40 early promoter and the long terminal repeat of human T cellleukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol.8:466-472).

In addition to the antibody chain genes and regulatory sequences,recombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies described herein in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13). Antibodies of the present inventioncan also be produced in glycoengineered strains of the yeast Pichiapastoris. Li et al. (2006) Nat. Biotechnol. 24:210.

Preferred mammalian host cells for expressing the recombinant antibodiesdescribed herein include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. (USA) 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

The N- and C-termini of antibody polypeptide chains of the presentinvention may differ from the expected sequence due to commonly observedpost-translational modifications. For example, C-terminal lysineresidues are often missing from antibody heavy chains. Dick et al.(2008) Biotechnol. Bioeng. 100:1132. N-terminal glutamine residues, andto a lesser extent glutamate residues, are frequently converted topyroglutamate residues on both light and heavy chains of therapeuticantibodies. Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al.(2011) J. Biol. Chem. 286:11211.

VII. Assays

Antibodies described herein can be tested for binding to CD40 by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified CD40 at 1-2 μg/ml in PBS, and then blocked with 5% bovine serumalbumin in PBS. Dilutions of antibody (e.g., dilutions of plasma fromCD40-immunized mice) are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween and then incubated withsecondary reagent (e.g., for human antibodies, or antibodies otherwisehaving a human heavy chain constant region, a goat-anti-human IgGFc-specific polyclonal reagent) conjugated to horseradish peroxidase(HRP) for 1 hour at 37° C. After washing, the plates are developed withABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by aspectrophotometer at OD 415-495. Sera from immunized mice are thenfurther screened by flow cytometry for binding to a cell line expressinghuman CD40, but not to a control cell line that does not express CD40.Briefly, the binding of anti-CD40 antibodies is assessed by incubatingCD40 expressing CHO cells with the anti-CD40 antibody at 1:20 dilution.The cells are washed and binding is detected with a PE-labeledanti-human IgG Ab. Flow cytometric analyses are performed using aFACScan flow cytometry (Becton Dickinson, San Jose, CA). Preferably,mice that develop the highest titers will be used for fusions. Analogousexperiments may be performed using anti-mouse detection antibodies ifmouse anti-huCD40 antibodies are to be detected.

An ELISA as described above can be used to screen for antibodies and,thus, hybridomas that produce antibodies that show positive reactivitywith the CD40 immunogen. Hybridomas that produce antibodies that bind,preferably with high affinity, to CD40 can then be subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can then be chosen for makinga cell bank, and for antibody purification.

To purify anti-CD40 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, NJ).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-CD40 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, IL). Biotinylated MAb binding canbe detected with a streptavidin labeled probe. Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using CD40 coated-ELISA plates as described above.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

To test the binding of monoclonal antibodies to live cells expressingCD40, flow cytometry can be used. Briefly, cell lines expressingmembrane-bound CD40 (grown under standard growth conditions) are mixedwith various concentrations of monoclonal antibodies in PBS containing0.1% BSA at 4° C. for 1 hour. After washing, the cells are reacted withPhycoerythrin (PE)-labeled anti-IgG antibody under the same conditionsas the primary antibody staining. The samples can be analyzed by FACScaninstrument using light and side scatter properties to gate on singlecells and binding of the labeled antibodies is determined. Analternative assay using fluorescence microscopy may be used (in additionto or instead of) the flow cytometry assay. Cells can be stained exactlyas described above and examined by fluorescence microscopy. This methodallows visualization of individual cells, but may have diminishedsensitivity depending on the density of the antigen.

Anti-huCD40 antibodies can be further tested for reactivity with theCD40 antigen by Western blotting. Briefly, cell extracts from cellsexpressing CD40 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.IgG binding can be detected using anti-IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,MO).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-CD40 antibodies include standard assays knownin the art, for example, Biolayer Interferometry (BLI) analysis, andBIACORE surface plasmon resonance (SPR) analysis using a BIACORE 2000SPR instrument (Biacore AB, Uppsala, Sweden).

In one embodiment, an antibody specifically binds to the extracellularregion of human CD40. An antibody may specifically bind to a particulardomain (e.g., a functional domain) within the extracellular domain ofCD40. In certain embodiments, the antibody specifically binds to theextracellular region of human CD40 and the extracellular region ofcynomolgus CD40. Preferably, an antibody binds to human CD40 with highaffinity.

VIII. Bispecific Molecules

Antibodies described herein may be used for forming bispecificmolecules. An anti-CD40 antibody, or antigen-binding fragments thereof,can be derivatized or linked to another functional molecule, e.g.,another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody describedherein may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific moleculedescribed herein, an antibody described herein can be functionallylinked (e.g., by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, antibody fragment, peptide or binding mimetic, suchthat a bispecific molecule results.

Accordingly, provided herein are bispecific molecules comprising atleast one first binding specificity for CD40 and a second bindingspecificity for a second target epitope. In an embodiment describedherein in which the bispecific molecule is multispecific, the moleculecan further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies thatcan be employed in the bispecific molecules described herein are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules described herein can be prepared by conjugatingthe constituent binding specificities using methods known in the art.For example, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. (USA) 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,IL).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific moleculedescribed herein can be a single chain molecule comprising one singlechain antibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed using art-recognized methods, such as enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,bioassay (e.g., growth inhibition), or Western Blot assay. Each of theseassays generally detects the presence of protein-antibody complexes ofparticular interest by employing a labeled reagent (e.g., an antibody)specific for the complex of interest.

IX. Compositions

Further provided are compositions, e.g., a pharmaceutical compositions,containing one or more anti-CD40 antibodies, or antigen-bindingfragment(s) thereof, as described herein, formulated together with apharmaceutically acceptable carrier. Such compositions may include oneor a combination of (e.g., two or more different) antibodies, orimmunoconjugates or bisplecific molecules described herein. For example,a pharmaceutical composition described herein can comprise a combinationof antibodies (or immunoconjugates or bispecifics) that bind todifferent epitopes on the target antigen or that have complementaryactivities.

In certain embodiments, a composition comprises an anti-CD40 antibody ata concentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, or at 1-300 mg/ml or 100-300 mg/ml.

Pharmaceutical compositions described herein also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-CD40 antibody described hereincombined with at least one other anti-cancer and/or T-cell stimulating(e.g., activating) agent. Examples of therapeutic agents that can beused in combination therapy are described in greater detail below in thesection on uses of the antibodies described herein.

In some embodiments, therapeutic compositions disclosed herein caninclude other compounds, drugs, and/or agents used for the treatment ofcancer. Such compounds, drugs, and/or agents can include, for example,chemotherapy drugs, small molecule drugs or antibodies that stimulatethe immune response to a given cancer.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds described herein may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition described herein also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents that delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsdescribed herein is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient that can be combined with a carrier materialto produce a single dosage form will generally be that amount of thecomposition that produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 0.01 percent to aboutninety-nine percent of active ingredient, preferably from about 0.1percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.A therapeutic antibody is usually administered on multiple occasions.Intervals between single dosages can be, for example, weekly, monthly,every three months or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody to the target antigen inthe patient. In some methods, dosage is adjusted to achieve a plasmaantibody concentration of about 1-1000 μg/ml and in some methods about25-300 μg/ml.

An antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan optionally be administered a prophylactic regime, although in manyimmune-oncology indications continued treatment is not necessary.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions described herein employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

A “therapeutically effective dosage” of an anti-CD40 antibody describedherein preferably results in a decrease in severity of disease symptoms,an increase in frequency and duration of disease symptom-free periods,or a prevention of impairment or disability due to the diseaseaffliction. In the context of cancer, a therapeutically effective dosepreferably prevents further deterioration of physical symptomsassociated with cancer. Symptoms of cancer are well-known in the art andinclude, for example, unusual mole features, a change in the appearanceof a mole, including asymmetry, border, color and/or diameter, a newlypigmented skin area, an abnormal mole, darkened area under nail, breastlumps, nipple changes, breast cysts, breast pain, death, weight loss,weakness, excessive fatigue, difficulty eating, loss of appetite,chronic cough, worsening breathlessness, coughing up blood, blood in theurine, blood in stool, nausea, vomiting, liver metastases, lungmetastases, bone metastases, abdominal fullness, bloating, fluid inperitoneal cavity, vaginal bleeding, constipation, abdominal distension,perforation of colon, acute peritonitis (infection, fever, pain), pain,vomiting blood, heavy sweating, fever, high blood pressure, anemia,diarrhea, jaundice, dizziness, chills, muscle spasms, colon metastases,lung metastases, bladder metastases, liver metastases, bone metastases,kidney metastases, and pancreatic metastases, difficulty swallowing, andthe like. Therapeutic efficacy may be observable immediately after thefirst administration of an agonistic anti-huCD40 mAb of the presentinvention, or it may only be observed after a period of time and/or aseries of doses. Such delayed efficacy my only be observed after severalmonths of treatment, up to 6, 9 or 12 months. It is critical not todecide prematurely that an agonistic anti-huCD40 mAb of the presentinvention lacks therapeutically efficacy in light of the delayedefficacy exhibited by some immune-oncology agents.

A therapeutically effective dose may prevent or delay onset of cancer,such as may be desired when early or preliminary signs of the diseaseare present. Laboratory tests utilized in the diagnosis of cancerinclude chemistries (including the measurement of soluble CD40 or CD40Llevels) (Hock et al. (2006) Cancer 106:2148; Chung & Lim (2014) J.Trans. Med. 12:102), hematology, serology and radiology. Accordingly,any clinical or biochemical assay that monitors any of the foregoing maybe used to determine whether a particular treatment is a therapeuticallyeffective dose for treating cancer. One of ordinary skill in the artwould be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

A composition described herein can be administered via one or moreroutes of administration using one or more of a variety of methods knownin the art. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies describedherein include intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition described herein can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules for use withanti-huCD40 antibodies described herein include: U.S. Pat. No.4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicamentsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-huCD40 antibodies described herein canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds described herein cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties that are selectively transported into specific cells or organs,thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J.Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais etal. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein Areceptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p 120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273.

X. Uses and Methods

The antibodies, antibody compositions and methods described herein havenumerous in vitro and in vivo utilities involving, for example,enhancement of immune response by agonizing CD40 signaling. In apreferred embodiment, the antibodies described herein are human orhumanized antibodies. For example, anti-huCD40 antibodies describedherein can be administered to cells in culture, in vitro or ex vivo, orto human subjects, e.g., in vivo, to enhance immunity in a variety ofdiseases. Accordingly, provided herein are methods of modifying animmune response in a subject comprising administering to the subject anantibody, or antigen-binding fragment thereof, described herein suchthat the immune response in the subject is enhanced, stimulated orup-regulated.

Preferred subjects include human patients in whom enhancement of animmune response would be desirable. The methods are particularlysuitable for treating human patients having a disorder that can betreated by augmenting an immune response (e.g., the T-cell mediatedimmune response). In a particular embodiment, the methods areparticularly suitable for treatment of cancer in vivo. To achieveantigen-specific enhancement of immunity, anti-huCD40 antibodiesdescribed herein can be administered together with an antigen ofinterest or the antigen may already be present in the subject to betreated (e.g., a tumor-bearing or virus-bearing subject). Whenantibodies to CD40 are administered together with another agent, the twocan be administered separately or simultaneously.

Also encompassed are methods for detecting the presence of human CD40antigen in a sample, or measuring the amount of human CD40 antigen,comprising contacting the sample, and a control sample, with a humanmonoclonal antibody, or an antigen binding fragment thereof, thatspecifically binds to human CD40, under conditions that allow forformation of a complex between the antibody or fragment thereof andhuman CD40. The formation of a complex is then detected, wherein adifference complex formation between the sample compared to the controlsample is indicative the presence of human CD40 antigen in the sample.Moreover, the anti-CD40 antibodies described herein can be used topurify human CD40 via immunoaffinity purification.

Given the ability of anti-huCD40 antibodies described herein to enhanceco-stimulation of T cell responses, e.g., antigen-specific T cellresponses, provided herein are in vitro and in vivo methods of using theantibodies described herein to stimulate, enhance or upregulateantigen-specific T cell responses, e.g., anti-tumor T cell responses.

CD4⁺ and CD8⁺ T cell responses can be enhanced using anti-CD40antibodies. The T cells can be Teff cells, e.g., CD4+ Teff cells, CD8+Teff cells, T helper (Th) cells and T cytotoxic (Tc) cells.

Further encompassed are methods of enhancing an immune response (e.g.,an antigen-specific T cell response) in a subject comprisingadministering an anti-huCD40 antibody described herein to the subjectsuch that an immune response (e.g., an antigen-specific T cell response)in the subject is enhanced. In a preferred embodiment, the subject is atumor-bearing subject and an immune response against the tumor isenhanced. A tumor may be a solid tumor or a liquid tumor, e.g., ahematological malignancy. In certain embodiments, a tumor is animmunogenic tumor. In certain embodiments, a tumor is non-immunogenic.In certain embodiments, a tumor is PD-L1 positive. In certainembodiments a tumor is PD-L1 negative. A subject may also be avirus-bearing subject and an immune response against the virus isenhanced.

Further provided are methods for inhibiting growth of tumor cells in asubject comprising administering to the subject an anti-huCD40 antibodydescribed herein such that growth of the tumor is inhibited in thesubject. Also provided are methods of treating chronic viral infectionin a subject comprising administering to the subject an anti-huCD40antibody described herein such that the chronic viral infection istreated in the subject.

In certain embodiments, an anti-huCD40 antibody is given to a subject asan adjunctive therapy. Treatments of subjects having cancer with ananti-huCD40 antibody may lead to a long-term durable response relativeto the current standard of care; long term survival of at least 1, 2, 3,4, 5, 10 or more years, recurrence free survival of at least 1, 2, 3, 4,5, or 10 or more years. In certain embodiments, treatment of a subjecthaving cancer with an anti-huCD40 antibody prevents recurrence of canceror delays recurrence of cancer by, e.g., 1, 2, 3, 4, 5, or 10 or moreyears. An anti-CD40 treatment can be used as a primary or secondary lineof treatment.

These and other methods described herein are discussed in further detailbelow.

Cancer

Provided herein are methods for treating a subject having cancer,comprising administering to the subject an anti-huCD40 antibodydescribed herein, such that the subject is treated, e.g., such thatgrowth of cancerous tumors is inhibited or reduced and/or that thetumors regress. An anti-huCD40 antibody can be used alone to inhibit thegrowth of cancerous tumors. Alternatively, an anti-huCD40 antibody canbe used in conjunction with another agent, e.g., other immunogenicagents, standard cancer treatments, or other antibodies, as describedbelow.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-huCD40antibody described herein, or antigen-binding fragment thereof. Theantibody may be a humanized anti-huCD40 antibody, a human chimericanti-huCD40 antibody, or a humanized non-human anti-huCD40 antibody,e.g., a human, chimeric or humanized anti-huCD40 antibody that competesfor binding with, or binds to the same epitope as, at least one of theanti-huCD40 antibodies specifically described herein.

Cancers whose growth may be inhibited using the antibodies of theinvention include cancers typically responsive to immunotherapy.Non-limiting examples of cancers for treatment include squamous cellcarcinoma, small-cell lung cancer, non-small cell lung cancer, squamousnon-small cell lung cancer (NSCLC), non NSCLC, glioma, gastrointestinalcancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, livercancer, colorectal cancer, endometrial cancer, kidney cancer (e.g.,renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractoryprostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, andhead and neck cancer (or carcinoma), gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastaticmalignant melanoma, such as cutaneous or intraocular malignantmelanoma), bone cancer, skin cancer, uterine cancer, cancer of the analregion, testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, solid tumors of childhood, cancer ofthe ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally-induced cancers including those induced by asbestos,virus-related cancers (e.g., human papilloma virus (HPV)-related tumor),and hematologic malignancies derived from either of the two major bloodcell lineages, i.e., the myeloid cell line (which produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cellline (which produces B, T, NK and plasma cells), such as all types ofleukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocyticand/or myelogenous leukemias, such as acute leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), andchronic myelogenous leukemia (CML), undifferentiated AML (M0),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphomas,T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-celllymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic(e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma/leukemia,mantle cell lymphoma, angio immunoblastic T-cell lymphoma, angiocentriclymphoma, intestinal T-cell lymphoma, primary mediastinal B-celllymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; andlymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,lymphoblastic lymphoma, post-transplantation lymphoproliferativedisorder, true histiocytic lymphoma, primary central nervous systemlymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL),hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia,diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma,diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma,precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC)(also called mycosis fungoides or Sezary syndrome), andlymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia;myelomas, such as IgG myeloma, light chain myeloma, nonsecretorymyeloma, smoldering myeloma (also called indolent myeloma), solitaryplasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL),hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;seminoma, teratocarcinoma, tumors of the central and peripheral nervous,including astrocytoma, schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoacanthoma,seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietictumors of lymphoid lineage, for example T-cell and B-cell tumors,including but not limited to T-cell disorders such as T-prolymphocyticleukemia (T-PLL), including of the small cell and cerebriform cell type;large granular lymphocyte leukemia (LGL) preferably of the T-cell type;a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-celllymphoma; cancer of the head or neck, renal cancer, rectal cancer,cancer of the thyroid gland; acute myeloid lymphoma, as well as anycombinations of said cancers. The methods described herein may also beused for treatment of metastatic cancers, refractory cancers (e.g.,cancers refractory to previous immunotherapy, e.g., with a blockingCTLA-4 or PD-1 antibody), and recurrent cancers.

An anti-huCD40 antibody can be administered as a monotherapy, or as theonly immunostimulating therapy, or it can be combined with animmunogenic agent in a cancer vaccine strategy, such as cancerous cells,purified tumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines (He et al. (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gp100, MAGEantigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF. Many experimental strategies forvaccination against tumors have been devised (see Rosenberg, S., 2000,Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62;Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D.2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCOEducational Book Spring: 730-738; see also Restifo, N. and Sznol, M.,Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997,Cancer: Principles and Practice of Oncology, Fifth Edition). In one ofthese strategies, a vaccine is prepared using autologous or allogeneictumor cells. These cellular vaccines have been shown to be mosteffective when the tumor cells are transduced to express GM-CSF. GM-CSFhas been shown to be a potent activator of antigen presentation fortumor vaccination. Dranoff et al. (1993) Proc. Natl. Acad. Sci. (USA)90: 3539-43.

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens. Rosenberg, S A (1999) Immunity 10: 281-7. In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. CD40 agonists can be used in conjunction witha collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). Tumor antigen can also be “neo-antigens” expressed incancer cells because of somatic mutations that alter protein sequence orcreate fusion proteins between two unrelated sequences (i.e., bcr-abl inthe Philadelphia chromosome), or idiotype from B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen that can be used in conjunction with CD40inhibition is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withCD40 agonism to activate (unleash) more potent anti-tumor responses.

Agonism of CD40 can also be combined with standard cancer treatments(e.g., surgery, radiation, and chemotherapy). Agonism of CD40 can beeffectively combined with chemotherapeutic regimes. In these instances,it may be possible to reduce the dose of chemotherapeutic reagentadministered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). Anexample of such a combination is an anti-huCD40 antibody in combinationwith decarbazine for the treatment of melanoma. Another example of sucha combination is an anti-huCD40 antibody in combination withinterleukin-2 (IL-2) for the treatment of melanoma. The scientificrationale behind the combined use of CD40 agonists and chemotherapy isthat cell death, that is a consequence of the cytotoxic action of mostchemotherapeutic compounds, should result in increased levels of tumorantigen in the antigen presentation pathway. Other combination therapiesthat may result in synergy with CD40 agonism through cell death areradiation, surgery, and hormone deprivation. Each of these protocolscreates a source of tumor antigen in the host. Angiogenesis inhibitorscan also be combined with CD40 agonists. Inhibition of angiogenesisleads to tumor cell death which may feed tumor antigen into host antigenpresentation pathways.

The anti-huCD40 antibodies described herein can also be used incombination with bispecific antibodies that target Fcα or Fcγreceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti-tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. The T cell arm of these responseswould be augmented by agonism of CD40. Alternatively, antigen may bedelivered directly to DCs by the use of bispecific antibodies that bindto tumor antigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofimmunosuppressive proteins expressed by the tumors. These include amongothers TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10(Howard & O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand(Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each ofthese entities can be used in combination with anti-huCD40 antibodies tocounteract the effects of the immunosuppressive agent and favor tumorimmune responses by the host.

Anti-CD40 antibodies are able to substitute effectively for T cellhelper activity. Ridge et al. (1998) Nature 393: 474-478. Activatingantibodies to T cell costimulatory molecules such as CTLA-4 (e.g., U.S.Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164:2160-2169), CD137/4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685(1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may alsoprovide for increased levels of T cell activation. Inhibitors of PD1 orPD-L1 may also be used in conjunction with anti-huCD40 antibodies.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-CD40 antibodies can increase the frequency and activityof the adoptively transferred T cells.

Chronic Viral Infections

In another aspect, the invention described herein provides a method oftreating an infectious disease in a subject comprising administering tothe subject an anti-huCD40 antibody, or antigen-binding fragmentthereof, such that the subject is treated for the infectious disease.

Similar to its application to tumors as discussed above,antibody-mediated CD40 agonism can be used alone, or as an adjuvant, incombination with vaccines, to enhance the immune response to pathogens,toxins, and self-antigens. Examples of pathogens for which thistherapeutic approach can be particularly useful, include pathogens forwhich there is currently no effective vaccine, or pathogens for whichconventional vaccines are less than completely effective. These include,but are not limited to HIV, Hepatitis (A, B, & C), Influenza, Herpes,Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. CD40 agonism is particularly useful against establishedinfections by agents such as HIV that present altered antigens over thecourse of the infections. These novel epitopes are recognized as foreignat the time of anti-human CD40 antibody administration, thus provoking astrong T cell response.

Some examples of pathogenic viruses causing infections treatable bymethods described herein include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods described herein include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lyme disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods described herein include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods described herein include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the above methods, CD40 agonism can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens. See, e.g., Holliger (1993)Proc. Natl. Acad. Sci. (USA) 90:6444-6448; Poljak (1994) Structure2:1121-1123.

Vaccine Adjuvants

Anti-huCD40 antibodies described herein can be used to enhanceantigen-specific immune responses by co-administration of an anti-huCD40antibody with an antigen of interest, e.g., a vaccine. Accordingly,provided herein are methods of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-huCD40 antibody, or antigen-binding fragmentthereof, such that an immune response to the antigen in the subject isenhanced. The antigen can be, for example, a tumor antigen, a viralantigen, a bacterial antigen or an antigen from a pathogen. Non-limitingexamples of such antigens include those discussed in the sections above,such as the tumor antigens (or tumor vaccines) discussed above, orantigens from the viruses, bacteria or other pathogens described above.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) described herein in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, anti-huCD40 antibodies described herein can beco-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immuno-complex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/ml doseonce every four weeks and adriamycin is intravenously administered as a60-75 mg/ml dose once every 21 days. Co-administration of anti-CD40antibodies, or antigen binding fragments thereof, described herein withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such co-administration can solve problems due to development ofresistance to drugs or a change in the antigenicity of the tumor cellsthat would render them unreactive with the antibody.

Also within the scope described herein are kits comprising the antibodycompositions described herein (e.g., human antibodies, bispecific ormultispecific molecules, or immunoconjugates) and instructions for use.The kit can further contain at least one additional reagent, or one ormore additional human antibodies described herein (e.g., a humanantibody having a complementary activity that binds to an epitope inCD40 antigen distinct from the first human antibody). Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or that otherwise accompanies the kit.

Combination Therapies

In addition to the combinations therapies provided above, anti-CD40antibodies described herein can also be used in combination therapy,e.g., for treating cancer, as described below.

The present invention provides methods of combination therapy in whichan anti-huCD40 antibody is co-administered with one or more additionalagents, e.g., antibodies, that are effective in stimulating immuneresponses to thereby further enhance, stimulate or upregulate immuneresponses in a subject.

Generally, an anti-huCD40 antibody described herein can be combined with(i) an agonist of another co-stimulatory receptor and/or (ii) anantagonist of an inhibitory signal on T cells, either of which resultsin amplifying antigen-specific T cell responses (immune checkpointregulators). Most of the co-stimulatory and co-inhibitory molecules aremembers of the immunoglobulin super family (IgSF), and anti-CD40antibodies described herein may be administered with an agent thattargets a member of the IgSF family to increase an immune response. Oneimportant family of membrane-bound ligands that bind to co-stimulatoryor co-inhibitory receptors is the B7 family, which includes B7-1, B7-2,B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5(VISTA), and B7-H6. Another family of membrane bound ligands that bindto co-stimulatory or co-inhibitory receptors is the TNF family ofmolecules that bind to cognate TNF receptor family members, whichinclude CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL,CD137/4-1BB, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4,OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL,BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR,EDA2, TNFR1, Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α 1β2,FAS, FASL, RELT, DR6, TROY, NGFR (see, e.g., Tansey (2009) DrugDiscovery Today 00:1).

In another aspect, anti-huCD40 antibodies can be used in combinationwith antagonists of cytokines that inhibit T cell activation (e.g.,IL-6, IL-10, TGF-β, VEGF; or other “immunosuppressive cytokines,” orcytokines that stimulate T cell activation, for stimulating an immuneresponse, e.g., for treating proliferative diseases, such as cancer.

The agonist anti-huCD40 antibodies and combination antibody therapiesdescribed herein may also be used in conjunction with other well knowntherapies that are selected for their particular usefulness against theindication being treated (e.g., cancer). Combinations of the agonistanti-huCD40 antibodies described herein may be used sequentially withknown pharmaceutically acceptable agent(s).

For example, the agonist anti-huCD40 antibodies and combination antibodytherapies described herein can be used in combination (e.g.,simultaneously or separately) with an additional treatment, such asirradiation, chemotherapy (e.g., using camptothecin (CPT-11),5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel,gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol),doxorubicin, 5-fu, or camptothecin+apo2l/TRAIL (a 6×combo)), one or moreproteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2inhibitors (e.g., BH3I-2′ (bcl-xl inhibitor), indoleamine dioxygenase-1(IDO1) inhibitor (e.g., INCB24360), AT-101 (R-(−)-gossypol derivative),ABT-263 (small molecule), GX-15-070 (obatoclax), or MCL-1 (myeloidleukemia cell differentiation protein-1) antagonists), iAP (inhibitor ofapoptosis protein) antagonists (e.g., smac7, smac4, small molecule smacmimetic, synthetic smac peptides (see Fulda et al., Nat Med 2002;8:808-15), ISIS23722 (LY2181308), or AEG-35156 (GEM-640)), HDAC (histonedeacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab),angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agentstargeting VEGF and VEGFR (e.g., AVASTIN), synthetic triterpenoids (seeHyer et al., Cancer Research 2005; 65:4799-808), c-FLIP (cellularFLICE-inhibitory protein) modulators (e.g., natural and syntheticligands of PPAR7 (peroxisome proliferator-activated receptor 7), 5809354or 5569100), kinase inhibitors (e.g., Sorafenib), trastuzumab,cetuximab, Temsirolimus, mTOR inhibitors such as rapamycin andtemsirolimus, Bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKTinhibitors, Lenalildomide, GSK3β inhibitors, IAP inhibitors and/orgenotoxic drugs.

The agonist anti-huCD40 antibodies and combination antibody therapiesdescribed herein can further be used in combination with one or moreanti-proliferative cytotoxic agents. Classes of compounds that may beused as anti-proliferative cytotoxic agents include, but are not limitedto, the following:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with agonistanti-huCD40 antibodies, without limitation, taxanes, paclitaxel(paclitaxel is commercially available as TAXOL™), docetaxel,discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones,epothilone A, epothilone B, epothilone C, epothilone D, epothilone E,epothilone F, furanoepothilone D, desoxyepothilone B1,[17]-dehydrodesoxyepothilone B, [18]dehydrodesoxyepothilones B,C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A,trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D,16-desmethylepothilone B, epothilone B10, discoderomolide, patupilone(EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (Discodermolide),TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), HalichondrinB, Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974,Cyrptophycins, LY-355703, Maytansinoid immunoconjugates (DM-1), MKC-1,ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731,STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with agonistanti-huCD40 antibodies described herein, hormones and steroids(including synthetic analogs), such as 17a-Ethinylestradiol,Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,Dromostanolone propionate, Testolactone, Megestrolacetate,Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone,Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine,Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEX™,can also be administered to the patient. When employing the methods orcompositions described herein, other agents used in the modulation oftumor growth or metastasis in a clinical setting, such as antimimetics,can also be administered as desired.

Methods for the safe and effective administration of chemotherapeuticagents are known to those skilled in the art. In addition, theiradministration is described in the standard literature. For example, theadministration of many of the chemotherapeutic agents is described inthe Physicians' Desk Reference (PDR), e.g., 1996 edition (MedicalEconomics Company, Montvale, N.J. 07645-1742, USA); the disclosure ofwhich is incorporated herein by reference thereto.

The chemotherapeutic agent(s) and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent(s) and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent(s) and/or radiation therapy on that disease.Also, in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents on the patient, and in view of the observed responsesof the disease to the administered therapeutic agents.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1

Generation of CD40/FcγR humanized mice

To generate an accurate and efficient model that can readily evaluatethe activity of human anti-CD40 Fc variants and enable the selection ofan optimized clinical candidate, we generated a unique mouse modelhumanized for CD40 and FcγRs. First, humanized CD40 mice on a mouseCD40-deficient background were generated. The expression pattern of thehuman CD40 BAC transgene on different immune cell populations in thesemice were evaluated and found that human CD40 expression on blood Bcells, dendritic cells, monocytes and macrophages, but not on T cell,neutrophils and NK cell populations is similar to the pattern found onhuman cells and their mouse orthologes (FIG. 1A).

The functionality of the huCD40 transgene in these mice, was verified byevaluating the formation of germinal centers (GC), a process thatrequires CD40 signaling (Basso et al. (2004) Blood 104, 4088-4096).While CD40^(−/−) mice lost the ability to form a GC, this phenotype wasrestored upon the introduction of the huCD40 transgene FIG. 1B, mediatedby the interaction of the huCD40 with the mouse CD40 ligand, which havesimilar binding kinetics and affinity to human CD40 ligand FIG. 2 .CD40^(−/−) mice have a deficient antigen-specific IgG response uponimmunization, which was restored upon introduction of the huCD40transgene FIG. 1C, confirming that hCD40 transgene functionallycomplements the mouse CD40 deficiency. Together, these data demonstratethat the humanized CD40 mice recapitulate the expression pattern andfunction of the human gene. To generate a mouse model in which fullyhuman agonistic IgGs against human CD40 can be evaluated, these CD40humanized mice were crossed to our previously described humanized FcγRmice (characterized in detail by (DiLillo and Ravetch (2015) Cell 161,1035-1045; Smith et al., 2012 PNAS (USA) 109, 6181-6186) resulting in astrain of mice expressing the human CD40 and alpha chains of the FCGR1A,FCGR2AR131, FCGR2BI232, FCRG3AF158, and FCGR3B genes under the controlof their endogenous human regulatory elements on an isogenic backgrounddeleted for the homologous mouse genes.

FcγRαnull mice have FcγR α chain deletion of Fcgr2b, Fcgr3, and Fcgr4,and are crossed to FcγRI−/− mice (Barnes et al., 2002 Immunity 16,379-389. They were generated in a C57BL/6 background and characterizedas previously described (Smith, et al. (2012) PHAS USA 109, 6181-6186).FcγR humanized mice (FcγRαnull, hFcγRI⁺, FcγRIIa^(R131+), FcγRIIb⁺,FcγRIIIa^(F58+), and FcγRIIIb⁺) generated and extensively characterizedas previously described (Smith, et al. (2012) PNAS (USA) 109,6181-6186). Human CD40 transgenic mice were generated on a C57BL6genetic background by pronuclear injection of linearized RP11-177B15 BACDNA (Osoegawa, et al. (2001) Genome Research 11, 483-496) and were matedwith CD40-knockout (“CD40^(−/−)”) mice (The Jackson Laboratory) toobtain CD40^(−/−)huCD40^(+/+) mice. CD40^(−/−)huCD40^(+/+) mice weremated with FcγR humanized mice to obtain the humanized CD40 and FcγRmice (referred “hCD40/FcγR”) containing theCD40^(−/−)hCD40⁺Fcgra^(−/−)Fcgr1^(−/−)hFCGRI⁺hFCGRIIA⁺hFCGRIIB⁺hFCGRIIIA⁺hFCGRIIIB⁺ genotype.hCD40/hFcRIIB⁺/hFcRIIA⁺ and hCD40/hFcRIIB⁺/hFcRIA⁻ mice described inFIG. 4D were obtained during the mating described for the generation ofhCD40/FcγR mice.

Example 2 OVA-Specific T Cell Response

Mice were immunized through i.p. injection with 2 μg ofDEC-OVA(mIgG1-D265A) (produced as previously described by (Li andRavetch (2011) Science 333, 1030-1034) in the presence or absence of 10μg of anti-CD40 IgGs (except of ChiLob IgGs that were used at 40μg/mice) with one of the various Fc's. Seven days later peripheral bloodwas collected and stained with FITC-conjugated anti-CD4, APC-conjugatedanti-CD8a and PE-conjugated OVA peptide SIINFEKL H-2^(b) tetramer(tet-OVA, Beckman Coulter) and analyzed on BD LSRForttesa.

Example 3 Flow Cytometry

Cell populations were defined by the following markers; DC (human:HLA-DR⁺BDCA1⁺CD209⁺CD3⁻CD14⁻CD19⁻CD59⁻; mouse: CD11b⁺CD11c⁺MHCII⁺F4/80⁻), monocytes (human: CD14⁺HLA-DR⁺CD15⁻; mouse:CD11b⁺Ly6C⁺F4/80⁻CD11c⁻), macrophages (human: CD14⁺CD68+; mouse:CD11b+F4/80+Ly6C-Ly6G-), B cells (human: CD19+; mouse: B220+), T cells(human: CD3⁺CD56⁻; mouse: CD3), NK cells (human: CD16⁺CD56⁺CD3⁻; mouse:NK1.1), neutrophils (human: CD15⁺CD16⁺CD49d⁻; mouse:CD11b⁺Ly6G⁺Ly6C^(int)F4/80).

Example 4 H1N1 Immunization

Mice were immunized with recombinant influenza H1N1 (Sino BiologicalInc.) in the presence of Alum as adjuvant. After 11 days blood wascollected and analyzed for anti-influenza H1N1-specific IgG usingstandard ELISA protocol.

Example 5 SPR

All experiments were performed with a Biacore T100 surface plasmonresonance (SPR) system (Biacore, GE Healthcare), as previously described(Bournazos, et al., 2014, Cell 158, 1243-1253). Briefly, experimentswere performed at 25-C in HBS-EP+ buffer (10 mM HEPES, pH 7.4; 150 mMNaCl; 3.4 mM EDTA; 0.005% (v/v) surfactant P20). For the measurement ofthe affinity of IgG subclass variants for FcγRs and CD40 recombinantIgGs were immobilized on Series S CM5 chips by amine coupling andsoluble ectodomains of FcγRs or CD40 samples were injected through flowcells at different concentrations. For some FcγRs, the measurements wererepeated in a reversed orientation while immobilizing the FcγR andinjecting soluble IgGs. Background binding to blank immobilized flowcells was subtracted and affinity constants were calculated usingBIAcore T100 evaluation software (GE Healthcare) using the 1:1 Langmuirbinding model.

Example 6 SPR-Based Competition Assay

SPR competition experiments were performed on a Biacore T100 instrumentusing a running buffer of 10 mM sodium phosphate, 130 mM sodiumchloride, 0.05% Tween 20, pH 7.1 at 25° C., on a surface consisting ofhCD40-Fc immobilized on a CM5 sensor chip using standard amine couplingchemistry. Competition for binding to hCD40L-Fc was assessed using the“dual injection” function in the T100 control software, by injectingmolecule 1 (parental antibody or CD40L), immediately followed by thesame concentration of molecule 1, or a mixture of molecule 1 plusmolecule 2. Binding responses were compared to a control injection ofmolecule 2 alone. All experiments were performed using 180 s associationand dissociation times at 30 μl/min. The surface was successfullyregenerated between cycles using two 15 s pulses of 10 mM glycine pH 1.5at a flow rate of 30 μl/min.

Example 7 CD40 Binding ELISA

Binding specificity and affinity of IgG subclasses were determined byELISA using recombinant CD40 (Sino Biological Inc.). ELISA plates (Nunc)were coated overnight at 4C with recombinant extracellular domain ofhuman CD40 (1 μg/well). All sequential steps were performed at roomtemperature. After being washed, the plates were blocked for 1 hr withPBS/2% skim milk and were subsequently incubated for 1 h with seriallydiluted IgGs (1:3 consecutive dilutions in PBS/2% skim milk). Afterwashing, plates were incubated for 1 hr with HRP-conjugated anti-humanIgG (Jackson ImmunoResearch). Detection was performed usingtwo-component peroxidase substrate kit (KPL) and reactions stopped withthe addition of 1 M phosphoric acid. Absorbance at 405 nm wasimmediately recorded using a SpectraMax Plus spectrophotometer(Molecular Devices) and background absorbance from negative controlsamples was subtracted.

Example 8 Generation and Production of Anti-CD40 Fc Variants

The variable heavy and light regions of anti-human CD40 Ab clone 21.4.1(U.S. Pat. No. 7,338,660) were synthesized (Genwize) and cloned intomammalian expression vectors with human IgG1, human IgG2, or human kappaFc backbones, as previously described (Li and Ravetch (2011) Science333, 1030-1034). For the generation of Fc domain variants of human IgG1(N297A, S267E, S267E/L328F, G237D/P238D/P271G/A330R,G237D/P238D/H268D/P271G/A330R) and human IgG2 (C127S, C232S),site-directed mutagenesis using specific primers was performed based onthe QuikChange site-directed mutagenesis Kit II (Agilent Technologies)according to manufacturer's instructions. Mutated plasmid sequences werevalidated by direct sequencing (Genewiz).

Antibodies were generated by transient transfection of HEK293T cells(ATCC), purified using Protein G Sepharose 4 Fast Flow (GE Healthcare),dialyzed in PBS, and sterile filtered (0.22 mm), as previously described(Nimmerjahn, et al. (2005) Immunity 23, 41-51). Purity was assessed bySDS-PAGE and Coomassie staining, and was estimated to be >90%. Abs usedfor in vivo experiments were quantified for endotoxin (LPS)contamination by the Limulus Amebocyte Lysate (LAL) assay and verifiedto have levels<0.1 EU/μg. Polyclonal human IgG was were purchased fromBio X Cell.

Example 9 Tumor Challenge and Treatment

MC38 cells (2×10⁶) were implanted subcutaneously and tumor volumes weremeasured every 2-3 days with an electronic caliper and reported asvolume using the formula (L₁ ²×L₂)2, whereas L₁ is the shortest diameterand L₂ is the longest diameter. 7 days after tumor inoculation, micewere randomized by tumor size (day 0) and received intraperitoneal (i.p)injection of 200 μg anti-CD40 or control IgGs. Mice received anadditional 200 μg of IgG treatment at days 3. For the B16 lungmetastasis model, mice were injected intravenously with 1×10⁶ B16-F10cells and treated with 40 μg of the indicated Abs on days 1 and 4 aftertumor cell injection. On day 14 lungs were harvested and analyzed forthe presence of surface metastasis foci by using a dissectingmicroscope.

Example 10 Generation of Human Anti-CD40-Fc-Variant Clinical Candidates

To test whether the in vivo activity of human IgGs targeting human CD40requires interaction with huFcγRIIB and determine if such interactionscan be further engineered to optimize the activity of the parentantibody, the variable regions of anti-CD40 clone 2141 (CP-870,893,originally an IgG2 isotype) were cloned into Fc-modified Abs withdifferential capacity to engage human FcγRs. These include wild typehuman IgG1 and a series of mutated IgG1s with increased bindingaffinities to hu FcγRIIB. ELISA FIG. 1D and SPR (Table 3) confirmed thatthe different Fc domains introduced into 2141 Abs did not alter eithertheir binding specificity or their affinity to human CD40. Table 4summarizes the affinities of different Fc variants of clone 2141 torecombinant huFcγRI, hFcγRIIA, huFcγRIIB, and huFcγR IIIA, as evaluatedby SPR.

TABLE 3 Affinities of 21.4.1 Fc variants to human CD40 Fc Variant Ka(1/Ms) Kd (1/s) KD (M) IgG1 5.973*10⁴ 1.189*10⁻³ 1.991*10⁻⁸ IgG1-N297A5.234*10⁴ 1.161*10⁻³ 2.219*10⁻⁸ IgG1-S267E 4.999*10⁴ 1.155*10⁻³2.309*10⁻⁸ IgG1-S267E/L328F 4.232*10⁴ 1.159*10⁻³ 2.738*10⁻⁸IgG1-G237D/P238D/P271G/ 4.217*10⁴ 1.129*10⁻³ 2.678*10⁻⁸ A330R (V9)IgG1-G237D/P238D/H268D/  4.1*10⁴ 1.125*10⁻³ 2.743*10⁻⁸ P271G/A330R (V11)IgG2 3.486*10⁴ 1.081*10⁻³ 3.101*10⁻⁸ IgG2-C232S (IgG2A) 3.515*10⁴1.088*10⁻³ 3.095*10⁻⁸ IgG1-C127S (IgG2B) 3.549*10⁴ 1.096*10⁻³ 3.088*10⁻⁸

Binding constants were obtained by SPR analysis with immobilized IgGsand soluble CD40.

TABLE 4 Affinities of 21.4.1 Fc variants to human FcγRs InhibitoryActivating hFcyRIIB hFcyRI hFcyRIIA^(R131) hFcyRIIIA^(F158) Fc variantK_(D) (M) Fold K_(D) (M) Fold K_(D) (M) Fold K_(D) (M) Fold IgG 3.01 ×10⁻⁶ 1 5.17 × 10⁻⁹ 1 1.16 × 10⁻⁶ 1 6.7 × 10⁻⁶ 1 wild type N297A n.d.b NAn.d.b NA n.d.b NA n.d.b NA SE 8.33 × 10⁻⁸ 30.2  2.6 × 10⁻⁹ 1.1  9.8 ×10⁻⁸ 15.8 n.d.b NA SELF 4.31 × 10⁻⁸ 69.8 3.68 × 10⁻⁹ 1.4 1.77 × 10⁻⁸65.5 n.d.b NA V9 9.39 × 10⁻⁸ 32 5.78 × 10⁻⁷ 0.009 4.11 × 10⁻⁶ 0.28 n.d.bNA V11 3.15 × 10⁻⁸ 95.6  2.3 × 10⁻⁷ 0.022 3.84 × 10⁻⁶ 0.3 n.d.b NA IgG2<10⁻⁵ NA n.d.b NA <10⁻⁵ NA <10⁻⁵ NA Binding constants were obtained bySPR analysis. Fold = K_(D)(IgG1)/K_(D)(Fc variant) n.d.b, no detectablebinding; NA, not applicable. K_(D) values and fold changes compared towild type IgGI for the SE mutant are from references (smith et al andChu et al 2008).

Example 11

FcγR-Engagement is Required for the In Vivo Activity of Human Anti-CD40mAbs

While the IgG1 isotype has relatively high affinity interactions withall human FcγRs, the IgG2 isotype of CP-870,893 has very low bindingaffinities to human FcγRs, with the exception of the FcγRIIAH131 (FIG.3A and Table 4). The efficacy of the IgG1 and IgG2 isotypes of anti-CD40in vivo in the context of human FcγRs, was compared by testing theirability to activate and expand T cells in the humanized CD40/FcγR model.Ovalbumin (OVA) was delivered to dendritic cells by the chimericanti-DEC205 Ab conjugated to OVA (“DEC-OVA” (Li and Ravetch (2011)Science 333, 1030-1034)) together with either human IgG1-Fc, IgG2-Fc, orN297A Fc-null subclasses of CP-870,893 anti-CD40 mAb, and monitored forthe presence of OVA-specific T cells in the circulation after 7 days(FIG. 3B). While both IgG1 and IgG2 isotypes had adjuvant effects on Tcell activation, IgG1 resulted in a significantly higher T cell responseas compared to the IgG2 isotype of the same anti-CD40 clone. Theactivity of IgG1 was completely reversed by introducing the N297Amutation, which prevents binding to FcγRs, implying that FcγR-engagementis required for the agonistic activity of the anti-CD40 IgG1. Thesignificant activity of IgG2 was lost when tested as deglycosylated formthat has reduced binding affinity to FcγRIIb compared to wild type IgG2.This implies on FcγR-dependent activity of anti-CD40 IgG2 as well andsuggesting that the relatively reduced activity of IgG2 compared to IgG1isotype may be explained by its low binding affinities to human FcγRs.In contrast to that conclusion, the agonistic activity of an anti-CD40antibody of the IgG2 subclass has been proposed to be FcγR-independentand the result of the unique configuration of the IgG2 hinge (White, etal. (2015) Cancer cell 27, 138-148), mediated by shuffled disulfidebonds between the IgG2 hinge and CH1 regions. To test that possibility,specific cysteines of CP-870,893 were mutated, which resulted in lockedconformational forms-the classical Y conformation “IgG2-A” and the morecompact conformation “IgG2-B” obtained by C232S and C127S mutations,respectively (Allen et al., 2009, Biochemistry 48, 3755-3766). Bothforms of IgG2 resulted in activity similar to wild type IgG2, implyingthat the hinge region conformation does not dictate the in vivoagonistic activity of the IgG2 isotype of this Ab clone in the contextof human FcγRs. Moreover, the in vivo agonist activity of the IgG2-A andIgG2-B forms of CP-870,893 in human CD40 transgenic mice on either amouse or human FcγR background were compared and found that in the mouseFcγR background only the IgG2-B form is active while in the human FcγRbackground both forms have significant and similar activity. The dataobtain for the CP-870,893 Abs is supported further by the similarhierarchy in agonistic activity observed for the IgG1 and the 2A and 2Bforms of IgG2 subclass of ChiLob 7/4, another agonistic human CD40 Abclone which recognizes an epitope distinct from 2141. While the IgG2subclass of ChiLob 7/4 was reported to have superior potency compared toits IgG1 form in the absence or presence of mouse FcγRs, we alsoobserved that for this clone, in the presence of human FcγRs, the IgG1subclass is superior to IgG2, and that both IgG2 conformational formshave similar activity. As observed for CP-870,893, when tested inhuCD40/mFcγR mice, only the IgG2-B form is active and results insignificant enhanced activity as compared to IgG2-A.

These data indicate that in the physiological context of human FcγRs,agonistic, human anti-CD40 IgGs depend on FcγR-engagement but not ontheir hinge-conformation for their in vivo activity. Importantly, thedata highlights the advantage of using the humanized FcγR mouse model inorder to appropriately study human IgGs activity. By considering theinteraction of human IgG with human FcγRs, these mice avoid theconfounding results that can be generated by using human IgGs in modelscarrying mouse FcγRs.

Example 12

Optimized activity of anti-CD40 human IgGs achieved by Fc variantsenhanced for FcγRIIB- but not FcγR HA-binding

We next determined whether increasing the binding interactions betweenhuman anti-CD40 antibodies and hFcγRIIB will result in increased in vivoefficacy. The binding affinity and selectivity of human IgGs to hFcγRIIBcan be increased by mutagenesis of their Fc domain. Fc variants ofCP-870,893 carrying the point mutations S267E (SE) and S267E/L328F(SELF) (Chu et al. (2008) Molecular Immunology 45, 3926-3933) resultwith 30- and 70-fold increased binding affinity to hFcγRIIB,respectively FIG. 4A and Table 4). When administrated to the humanizedFcγR/CD40 mice, these Fc variants resulted in small but significantincreases in their ability to activate T cells in vivo as compared toboth the wild type IgG1 and IgG2 variants of CP-870,893 FIG. 4B).

Due to the sequence and structural similarity between hFcγRIIA andhFcγRIIB, the SE and SELF mutations also result in increased bindingaffinity to the activating hFcγRIIA. Therefore, despite an increase intheir binding affinity to the inhibitory hFcγRIIB, the FcγRIIA/FcγRIIBbinding affinity ratio of these mutated IgGs is similar to that of wildtype IgG1 and were thus predicted to result with limited increasedactivity of this subclass as was observed FIG. 4B. To optimize theFc-engagement of FcγRIIB in the absence of FcγRIIA, we generated Fcvariants of CP-870,893 with the recently described mutations,G237D/P238D/P271G/A330R (V9) and G237D/P238D/H268D/P271G/A330R (V11),which enhance binding affinity specifically to hFcγRIIB but not tohFcγRIIA (Mimoto et al., (2013) PEDS 26, 589-598). V9-CP-870,893 andV11-CP-870,893 Fc variants result with 32- and 97-fold increased bindingaffinity to hFcγRIIB and about 3-fold decreased binding affinity tohFctγRIIA^(R)131 FIG. 4A and Table 4). Both V9 and V11 Fc variants havesignificantly improved in vivo activity compared to the the IgG2subclass of CP-870,893 (IgG2), and its SE-, and SELF-Fc variantsenhanced for both hFcγRIIB and hFcγRIIA. The 2141-V11 variant results in25-fold increase in T cell activation compared to CP-870,893-IgG2, and5-fold increase compared to the activity obtained by the SELF variantFIG. 4B. A similar hierarchy was observed for CP-870,893 Fc variantswhen change in body weight was determined after antibody administrationFIG. 5A. While all Fc variants tested resulted in statisticallysignificant decreases in body weight after a single injection ofanti-CD40, the group injected with V11-CP-870,893 had the mostsignificant reduction.

We analyzed the pharmacokinetic (PK) properties of these Fc variants totest whether their differential FcγR binding leads to differential PKclearance rates that can account for their differential agonisticactivities. SELF and V11 Fc variants of CP-870,893 were found to have afaster clearance rate than the IgG2 subclass (FIG. 4B), presumably aresult of their enhanced FcRIIB binding activity. However, despite thefact that SELF and V11 have faster clearance rates, they displaysuperior agonistic activity compared to IgG2. Similarly, despite thesimilar PK properties of SELF and V11, V11 is a superior agonistcompared to SELF. It is therefore excluded that the possibility thatdifferent PK properties of these Fc variants account for their agonisticactivity.

The influence of hFcγRIIA-engagement on the activity of CP-870,893, wasevaluated by comparing its activity in mice transgenic for human CD40and FcγRIIB, but not for FcγRIIA or mice transgenic to human CD40,FcγRIIB, and FcγRIIA (FIG. 4C). The T cell activation potency ofCP-870,893-IgG2 is significantly enhanced in the absence of FcγRIIA,indicating the negative role of FcγRIIA-engagement on the agonisticactivity of this anti-CD40 mAb.

The invention demonstrates that engineering CP-890,873 for enhancedhFcγRIIB-engagement while keeping a low FcγRIIA/FcγRIIB binding ratioresults in optimized in vivo agonistic activity of human anti-CD40 IgGs.

Increased immunostimulatory activity by selective enhancement of FcγRIIB binding was demonstrated for 2141 (CP-870,893) mAb that does notblock the binding of hCD40 to CD40L, demonstrating that agonist, humananti-CD40 mAbs can be optimized by selective enhancement ofFcγRIIB-binding through Fc engineering independent of their bindingepitopes and their ability to cross block CD40L binding to CD40.

The importance of FcγR interactions for the most potent human CD40 mAbwas demonstrated in the clinic, among others, and how selectivemanipulation of FcγR-interactions by Fc-engineering enhances the potencyof these drugs. These conclusions were made possible through the use ofa representative in vivo model that faithfully recapitulates thediversity and cell type specificity of the human FcγR system.

This invention also refutes the notion that the epitope specificity ofagonistic CD40 mAb determines its FcγR-requirements (FcγR-independentvs, -dependent, respectively) for activity. CP-870,893, ChiLob 7/4 doesnot compete with CD40L binding, but proved to be FcγR-dependent fortheir optimal activity in vivo.

Different modes-of-action can be observed between different mAb clonesalthough they share the same target molecule. For example, it wasrecently observed that antagonistic PD-1 mAbs have the potential todeliver FcγRIIB-dependent agonism based on their epitope specificity(Dahan et al. (2015) Cancer cell 28, 285-295). Although mouse models canbe very informative for evaluating mAb activity, translating of mAbactivity in the mouse to the human therapeutic is not straightforwardand therapeutic mechanisms observed for a mouse mAb can be altered whiledeveloping the homologous human IgGs. By humanizing both CD40 and FcγRs,a mouse model was generated that enables in vivo evaluation of clinicalproducts by considering the activity mediated by both their Fab and Fcdomains. These mice allow for “lead” selection among a panel of humanCD40 mAbs based on their in vivo agonistic potency. A similar approachshould be used for the generation of mice humanized for othertherapeutic targets on the huFcγR background for optimal selection ofadditional immunomodulatory mAbs.

Although improved agonistic activity is mediated by Fc variants enhancedfor both FcγRIIA and FcγRIIB binding (e.g, by S267E or S267E/L328F Fcmutations), their potency is limited by their enhanced binding to theactivating FcγR IIA. Therefore, Fc variants with selective enhancementin binding solely to the inhibitory FcγRIIB have been indicated by thisstudy as the most potent CD40 mAb derivatives. Lack of activity of mouseCD40 mAbs carrying the IgG2a subclass that preferentially bindsactivating FcγRs is associated with depletion of CD40 expressing cells(Li and Ravetch (2011) Science 333, 1030-1034). Since human FcγRIIIA,but not FcγR IIA, mediates in vivo depletion by mAbs (DiLillo andRavetch (2015) Cell 161, 1035-1045), and the S267E or S267E/L328Fmutants are enhanced to FcγRIIA but not FcγRIIIA, the reduced potency ofthe CD40 mAb mediated by FcγRIIA-engagement is not through depletion ofCD40 expressing cells. The mechanism that accounts for this inhibitoryeffect by FcγRIIA is the subject of our ongoing investigations.Histidine (H)/arginine (R) polymorphism at position 131 of FcγRIIAdictates its binding affinity to IgG2, FcγRIIa^(H131) has about 5-timeshigher affinity to IgG2 than FcγRIIa¹³¹ (van Sorge et al. (2003) TissueAntigens 61, 189-202). Due to the inhibitory effect of FcγRIIA on theactivity of CP-870,893, patients carrying the FcγRIIA^(131H/H) genotypemay have a reduced response to CP-870,893 treatment. Humanized micecarry the FcγRIIA^(131R/R) genotype but an FcγRIIA^(131H/H) strain isbeing generated so that the contribution of FcγRIIA allele polymorphismto the activity of anti-CD40 mAbs can be elucidated.

Example 13 V11 Fc Variant of Anti-CD40 Ab has Superior Anti-TumorActivity

Whether the increased agonistic activity observed for theFcγRIIB-enhanced mutated Fc variants can be translated into increasedanti-tumor activity of anti-CD40 mAbs was evaluated. Humanized CD40/FcγRmice were inoculated with the syngeneic MC38 colon adenocarcinoma tumorsand treated with 2141-IgG2, -SELF, and -V11 Fc variants of 2141anti-CD40 agonistic mAb (FIG. 6A). Treatments with both IgG2(CP-870,893) and SELF Fc variants results in similar antitumor effects(about 65% reduction in tumor volume compared to untreated control, and20% to 33% of tumor free mice, respectively). However, treatment withthe V11 Fc variant results in a dramatically improved antitumor responseand complete recovery from tumors of all the mice in that group. Asimilar trend was observed using the B16 metastatic melanoma model inwhich a statistically significant reduction in the number of lungmetastases was observed only in mice treated with V11 but not with SELFor IgG2 Fc variants FIG. 6B. These data indicate that the antitumoractivity of CP-870,893 can be enhanced by Fc engineering of the antibodyto provide selective enhancement of FcγRIIB-engagement and highlight theV11 Fc variant of this mAb clone as the optimal clinical candidate.

The unique hinge conformation of the human IgG2 isotype has recentlyreported to enhance the agonistic activity of CD40 mAbs in anFcγR-independent manner. It was therefore suggested that the superioragonistic activity observed for CP-870,893 is due to its IgG2 isotypecompared the IgG1 isotype of the other agonistic CD40 Ab in clinicalevaluations, ChiLob 7/4 and SGN40. When ChiLob 7/4 and SGN40 weregenerated as IgG2, they resulted in enhanced potency compared to theiroriginal IgG1 isotype (White et al. (2015) Cancer cell 27, 138-148). Adrawback of that study is that the mAbs were evaluated only in thepresence (or absence) of mouse FcγRs and their isotype-dependent potencyin the correct context of human FcγRs was not evaluated. Here wedemonstrated that using ChiLob 7/4 as IgG2 results in reduced activitycompared to IgG1 in the context of human FcγRs. We further evaluated theactivity of IgG1 vs IgG2 subclasses, including the 2A and 2B forms ofIgG2, of both CP-870,893 and ChiLob 7/4 and found that IgG1 is morepotent than IgG2 and its activity is FcγR-dependent. We thereforeconclude that the superior agonistic activity of anti-CD40 human IgG2observed in mice is not relevant to its clinical activity in humans.Moreover, the relatively high potency of CP-870,893 compared to theother CD40 mAbs is not a result to the IgG2 isotype and is likely theresult of the mAb recognition of a unique specific agonistic epitope.Finally, selective enhancement for FcγRIIb-binding is by far the mostefficient strategy to enhance the potency of CD40 mAb agonism.

TABLE 2 Summary of Sequence Listing SEQ ID Description  1Human CD40 (2141) Heavy Chain  2 Human CD40 Light Chain¹  3N297A heavy Chain  4 SE Heavy Chain  5 SELF Heavy Chain  6V9 Heavy Chain  7 V11 Heavy Chain  8 2141-IgG2-Heavy Chain  92141-IgG2 C127S-Heavy Chain 10 2141-IgG2 C232S-Heavy Chain 11human CD40 (NP_001241.1), 12 human CD40L (NP_000065.1) 13Signal Sequence SEQ ID NO: 1-2141-IgG1 Heavy chain

VS SATTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 2-2141-IgG1 Light chainDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 3-2141-IgG1 N297A Heavy chain

VS SATTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4-2141-IgG1 S267E-Heavy chain

VS SATTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 5-2141-IgG1 S267E/L328F-Heavy chain

VS SATTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 6-2141-IgG1-G237D/P238D/P271G/A330R  (V9)-Heavy chain

VS SATTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 7-2141-IgG1- G237D/P238D/H268D/P271G/ A330R (V11)-Heavy chain

VS SATTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 8-2141-IgG2-Heavy chain

SEQ ID NO: 9-2141-IgG2 C127S-Heavy chain

SEQ ID NO: 10-2141-IgG2 C232S-Heavy chain

SEQ ID NO: 13 Signal Sequence MVRLPLQCVLWGCLLTAVHP ¹The light chainsequences for all 2141 (CP-870, 893) Fc variants are identical to thesequence of SEQ Id NO: 2.

The Sequence Listing provides the sequences of the mature heavy andlight chains (i.e., sequences do not include signal peptides). A signalsequence for production of the antibodies of the present invention, forexample in human cells, is provided at SEQ ID NO: 13.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments disclosed herein. Such equivalents are intended to beencompassed by the following claims.

We claim:
 1. An isolated antibody, or antigen binding portion thereof,that (i) specifically binds to human CD40 (ii) comprises a mutant Fcregion having one or more mutations corresponding to one or moremutations in an IgG heavy chain selected from the group consisting ofSEQ ID Nos: 3-7, wherein the antibody competes for binding to human CD40with CP-870,893 or ChiLob,
 2141. 2. The antibody or antigen bindingportion thereof of claim 1 wherein the antibody has an enhancedspecificity of binding to FcγRIIb.
 3. The antibody or antigen bindingportion thereof of claim 2 exhibiting an A/I ratio of less than
 5. 4.The antibody or antigen binding portion thereof of claim 3 exhibiting anA/I ratio of less than
 1. 5. A nucleic acid encoding the heavy and/orlight chain variable region of the antibody, or antigen binding portionthereof, of claim
 1. 6. An expression vector comprising the nucleic acidof claim
 5. 7. A cell transformed with an expression vector of claim 6.8. A method of preparing an anti-human CD40 antibody, or antigen bindingportion thereof, comprising: a) expressing the antibody, or antigenbinding portion thereof, in the cell of claim 7; and b) isolating theantibody, or antigen binding portion thereof, from the cell.
 9. Apharmaceutical composition comprising: a) the antibody, or antigenbinding portion thereof, of claim 1; and b) a carrier.
 10. A method ofstimulating an immune response in a subject in need thereof comprisingadministering to the subject the pharmaceutical composition of claim 9.11. The method of claim 10, wherein the subject has a tumor and animmune response against the tumor is stimulated.
 12. The method of claim10, wherein the subject has a chronic viral infection and an immuneresponse against the viral infection is stimulated.
 13. A method oftreating cancer comprising administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical composition ofclaim
 9. 14. The method of claim 13, wherein the cancer is selected fromthe group consisting of: bladder cancer, breast cancer, uterine/cervicalcancer, ovarian cancer, prostate cancer, testicular cancer, esophagealcancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer,colon cancer, kidney cancer, head and neck cancer, lung cancer, stomachcancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,myeloma, sarcoma, and virus-related cancer.
 15. A method of treating achronic viral infection comprising administering to a subject in needthereof a therapeutically effective amount of the pharmaceuticalcomposition of claim 9.